Mass Spectrometric Analysis of Environmental

MASS SPECTROMETRIC ANALYSIS OF ENVIRONMENTAL

CONTAMINANTS, PROTEIN STRUCTURE AND EXPRESSION

IAN E. ATKINSON

Bachelor of Science in Zoology

Miami University

August 1996

Master of Science in Chemistry

Cleveland State University

May 2001

Submitted in partial fulfillment of requirement for the degree

DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY

at the

CLEVELAND STATE UNIVERSITY

DECEMBER, 2008

©COPYRIGHT BY IAN ERIC ATKINSON 2008

ii This dissertation has been approved

for the Department of CHEMISTRY

and the College of Graduate Studies by

______Dissertation Chairperson, Dr. Robert Wei

______Department & Date

______Dr. Lily Ng

______Department & Date

______Dr. John Masnovi

______Department & Date

______Dr. Xiang Zhou

______Department & Date

______Dr. Teresa Cutright

______Department & Date

iii Acknowledgements

This dissertation is dedicated to my loving parents, Linda & Les, without whose

encouragement and support none of this would have been possible. They have taken

every opportunity to enrich my life and education, and for that I am beyond thankful.

They are truly the most wonderful parents a son could ask for.

I would like to deeply thank my advisor, Dr Robert Wei. His direction, guidance and

patience have been fundamental in my progress and development as a scientist, a

researcher, and an educator. It has been an honor and a privilege working under his

tutelage, and I look forward to continuing our work for the good of society and the world.

I would like to express how very grateful I am for Dr Lily Ng’s involvement in my

life. Her wise counsel and insight has been fundamental in my education. Without her I would have never found my place here at Cleveland State University.

I would like to thank Dr Alan Riga and Dr Jerry Mundell, both of whom took a tremendous personal interest in my professional development and have helped prosper a wonderful outlook on science education and research within me. I owe so much of my holistic growth while at Cleveland State to these two gentlemen.

I would like to thank Dr Xiang Zhou for so many countless days of training and education on mass spectrometry. I will miss our conversations.

I would like to thank my family whose love and concern has motivated me to succeed. My brother David, sister-in-law Kristin, my niece Molly and nephew Colin, all my love, you are the light of my life. My Aunt Sondra, who helped me tremendously

with editing this work, my Uncle Joe, my Aunt Wendy, my Uncle Fred, my cousins

iv Aaron and Elliot, I cannot express how much your endless encouragement to follow my own path has meant to me.

I would like to express my humblest thanks to all of my friends, Adam, Amanda, Ann,

Bill, Brian, Carrie, Chad, Curt, Chamari, Deley, Eric, Jason, Jim, Jimmy, Jinhua, John,

Kate, Kerri, Lynn, Meg, Mike, Miles, Pam, Pat, Paul, Ram, Rachel, Robert, Brooke &

Tony, Isis & Ron, Kelly & Kevin, Meghan & Michael, Melissa & Michael, Nancy &

Dick, Nancy & Jeff, Rachel & Ray, Ruth & David, Sheila & Johnny, Wendy & David and many more that I do not have the room to mention here. I started this project in the hopes of taking direction of my dreams to reach a point wherein I could enact change upon the world. It has been my friends support and strength over the years that has held me up, kept my life full and allowed me to see this through.

I would also like to express my deep thanks and fullest respect to Mr. Ray Fogg. My mentor, role-model and dear friend, who was very much responsible in my choice of continuing my education and this path I have taken in life. I take the next steps with your inspiration and I follow in your footsteps to ensure tikkun olam.

v MASS SPECTROMETRIC ANALYSIS OF ENVIRONMENTAL

CONTAMINANTS, PROTEIN STRUCTURE AND EXPRESSION

IAN E. ATKINSON

ABSTRACT

There are a wide array of sciences at our disposal to further our understanding of the roles of toxins in the environment and their effects on living organisms. Of these disciplines, the field of mass spectrometry offers a powerful analytical means to identify

compounds in varying and complex sample media. The presence of altered expressions

of biomolecules upon exposure to toxins, the effects that these toxins may have on

biomolecule structure and the identity of toxins in the sample all can be determined by

mass spectrometry.

In this study mass spectrometric analysis has been applied to environmental

toxicological problems. First, the altered protein expression under various metal stress in

Helianthus annuus (dwarf sunflower), a proven hyperaccumulator of toxic metals was

analyzed by this research. Second, a model was developed for mass spectrometrically

determining the location and structural effects that another class of environmental toxins,

poly aromatic hydrocarbons (PAHs) can have on proteins. Third, the presence of PAHs

in environmental samples taken from Lake Bolgoda and Lake Beira, Sri Lanka, was

analyzed by mass spectrometry to determine the types, relative concentrations and

potential sources of these toxins.

The work has demonstrated the versatility and effectiveness of the methodology,

discovering novel protein expression in H. annuus upon metal exposure, pinpointing site

vi mediated adductions reactions on protein structure, and the qualitative and quantitative determination of toxins in environmental samples.

vii TABLE OF CONTENTS

ABSTRACT……………………..……………………………………………………… vi

TABLE OF CONTENTS…………………………………………………………….…viii

LIST OF TABLES…………………………………………………………………….…xii

LIST OF FIGURES…………...…………………………………………………….…..xiv

CHAPTER I LC ESI ION TRAP MS DETERMINATION OF ALTERED PROTEIN

EXPRESSION IN HELIANTHUS ANNUUS AFTER EXPOSURE TO HEAVY

METALS As, Cd, Cr, Ni AND Pb…………...……………………………...... …….1

1.1. Introduction…………….…………………………………………………………2

1.2. Methodology……………..…………………………………………………….. 13

1.2.1. Plant growth and exposure.………………..………………………………13

1.2.2. Protein extraction and gel separation…..……………...…..………………14

1.2.3. Sample preparation and tryptic digest…..……………………..………….14

1.2.4. LC separation…………………………….…………………..……………16

1.2.5. MS analysis……………………………….…………………..…………...17

1.2.6. MASCOT bioinformatics………………….………………..……………..17

1.3. Results……………….…………………………...……………………………..18

1.3.1. Expression of proteins in Sundance……………………..………………..18

1.3.1.1. Expression of proteins in response to arsenic and other metals in

Sundance…………………………………………………………………18

1.3.1.2. Expression of proteins in response to arsenic only in Sundance..24

1.3.1.3. Expression of proteins in response to lead and other metals in

viii Sundance………………………………………………………………...25

1.3.2. Expression of proteins in Teddy Bear…………………………….30

1.3.2.1. Expression of proteins in response to arsenic and other metals in

Teddy Bear………………………………………………………………30

1.3.2.2. Expression of proteins in response to chromium only in Teddy

Bear……………………………………………………………………....30

1.3.2.3. Expression of proteins in response to lead and other metals in

Teddy Bear………………………………………………………………31

1.3.2.4. Expression of proteins in response to lead and other metals 2D gel

separation in Teddy Bear………………………………………………..34

1.4. Discussion…………………………………………………………………...42

1.5. Conclusions………………………………………………………………….54

1.6. References…………………………………………………………………...58

CHAPTER II STUDY ON ADDUCTION REACTIONS IN CHYMOTRYPSIN’S

ACTIVE SITE…………………………………………………………………………...67

2.1. Background and Significance……………………………………………….68

2.2. Methodology………………………………………………………………...77

2.2.1. CHY adduct formation………………….……………………………77

2.2.1.1. CHY-TPCK adduct reaction……...……………………….…….77

2.2.1.2. Proposed CHY-PAH adduct reaction…………………………...77

2.2.2. Whole protein adduct analyses…………………….…………………78

2.2.2.1. LC separation and QQQ analysis……………….……………….78

2.2.2.2. LC separation and Ion Trap Analysis………………...…………79

ix 2.2.3. Hydrazinolysis analysis…………………………….………………...80

2.2.3.1. Hydrazinolysis…………………………………………………..80

2.2.3.2. LC separation and QQQ analysis……………………………….80

2.2.3.3. LC separation and Ion Trap analysis……………………………81

2.2.4. Enzymatic digest analysis……………………………………………83

2.2.5. Functional analysis of protein………………………………………..85

2.3. Results & Discussion

2.3.1. QQQ MS characterization of adduct in chymotrypsin………...…..…85

2.3.2. LC ESI-MS Ion characterization of adduct in chymotrypsin...…..…..90

2.3.3. UV spectroscopic analysis……………..…………………….……….92

2.3.4. Tryptic digest……………………………………..…………………..92

2.4. Conclusions………………………………………………………………….93

2.5. References…………………………………………………………………...95

CHAPTER III MS DETERMINATION OF POLYCYCLIC AROMATIC

HYDROCARBONS (PAHS) IN SEDIMENT AND WATER SAMPLES FROM

BOLGODA AND BEIRA LAKES……………………………………………….……..98

3.1. Abstract……………………………………………………………………...99

3.2. Introduction………………………………………………………………...101

3.3. Methodology……………………………………………………………….103

3.3.1. Sample site selection & collection……………...…………………...103

3.3.2. Sample preparation……………………………….…………………105

3.3.3. HPLC Sample analysis…………………………………..………….106

3.3.4. GCMS Sample analysis…………………………………..…………106

x 3.3.5. Evaluation of sediment toxicity…………………………..………...107

3.4. Results & Discussion………………………………………….…..………109

3.4.1. Beira Lake…………………………………………………………..109

3.4.2. Bolgoda Lake…………………………………………….………….113

3.4.3. PAH Ratios……………………………………………….…………116

3.4.4. Microtox Assay…………………………………………….………..119

3.5. References………………………………………………………………….121

APPENDICES……………………...…………………………………………………..124

A. Additional tables & figures for chapter 1……………...………………….125

B. Additional figures for chapter 2……………………………….....……….197

xi LIST OF TABLES

CHAPTER I

Table 1-I. 1-D Hydroponic Sundance Root As, Cd, Cr, Ni…………………………21

Table 1-II. 2-D Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb…………………….....37

CHAPTER II

Table 2-I. Secondary structure of chymotrypsin, individual amino acids molecular

weight and the molecular weight of the expected amino acid hydrazides.89

CHAPTER III

Table 3-I. Average PAH levels in Beira and Bolgoda Lakes……………………...110

Table 3-II. Ratios of two-three ring to four-five ring PAHs for Water and Sediment

samples from Beira and Bolgoda Lakes………………………………..117

Table 3-III. Phenanthrene/anthracene (P/A) and fluoranthene/pyrene (F/P) ratios for

water and sediment samples from Beira and Bolgoda Lakes…………..118

APPENDIX A

Table A-I. 1-D Hydroponic Sundance Leaf As only……………………………….129

Table A-II. 1-D Sundance Leaf Hydroponic Cd, Cr, Ni, Pb………………………..136

Table A-III. 1-D Sundance Stem Hydroponic Cd, Cr, Ni, Pb………………………..147

Table A-IV. 1-D Sundance Leaf Hydroponic (soluble fraction) Cd, Cr, Ni, Pb……..151

Table A-V. 1-D Sundance Leaf Soil 25-35 kDa EDTA samples Cd, Cr, Ni, Pb……156

Table A-VI. 1-D Teddy Bear Leaf Soil 25-35 kDa EDTA samples Cd, Cr, Ni, Pb…160

xii Table A-VII. 1-D Teddy Bear Leaf Soil Cr only……………………………………...163

Table A-VIII. 1-D Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb……………………...167

Table A-IX. 1-D Teddy Bear Stem Hydroponic Cd, Cr, Ni, Pb……………………..172

Table A-X. 1-D Teddy Bear Leaf Hydroponic (Soluble fraction) Cd, Cr, Ni, Pb...... 176

Table A-XI. 1-D Teddy Bear Leaf Hydroponic(Transmembrane fraction)Cd, Cr, Ni,

Pb……………………………………………………………………….179

Table A-XII. 2-D Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb……………………...181

xiii LIST OF FIGURES

CHAPTER I

Figure 1-1. Tandem mass spectra of polypeptide from Chitinase (Helianthus annuus)

as indicated (bold) in the primary amino acid sequence…………………20

Figure 1-2. Tandem mass spectra of peptide from an unknown protein type with high

sequence homology to pathogenesis-related protein 5-1 (Helianthus

annuus) as indicated (bold) in the primary amino acid sequence………..22

Figure 1-3. 1-D Gel Separation Sundance Hydroponic As…………………………..23

Figure 1-4. 2-D Gel Separation Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb………36

Figure 1-5. Tandem mass spectra of polypeptide from Chitinase (Helianthus annuus)

as indicated (bold) in the primary amino acid sequence…………………38

Figure 1-6. Tandem mass spectra of polypeptides from thaumatin-like protein

(Solanum tuberosum) as indicated (bold) in the primary amino acid

sequence………………………………………………………………….40

Figure 1-7. Semi-quantitative RT-PCR of total RNA samples showing an up-

regulation of Chitinase in samples containing As [11]…………………..43

Figure 1-8. Western Blot analysis of Sundance and Teddy Bear samples showing an

up-regulation of Chitinase in samples exposed to As [11]………………44

CHAPTER II

Figure 2-1. Conc. of Benzo [a] pyrene detected in air, soil, water, and plants and

food……………………………………..………………………………..68

Figure 2-2. Metabolism of Benzo [a] Pyrene……………………………………..….70

xiv Figure 2-3. Catalytic core of Chymotrypsin, a model Serine Peptidase……………...72

Figure 2-4. TPCK reaction/inhibition of CHY…………………………………….…73

Figure 2-5. Representative diagram of potential equilibrium reactions between an

electrophile E and neuleophilic residues N1 and N2………………….….75

Figure 2-6. QQQ Mass spectra of histidine and TPCK, optimization of small molecule

analysis…………………………………………………………..……….82

Figure 2-7. QQQ Mass spectra of TPCK and daughter analysis of parent molecule,

optimization of small molecule analysis CID……………………...…….83

Figure 2-8. LC-MS QQQ Whole CHY-TPCK (top) and CHY (bottom)………….…87

Figure 2-9. LC MS QQQ Separation of hydrazine digests of CHY-TPCK (top) and

CHY (bottom)……………………………………………………………88

Figure 2-10. LC MS QQQ hydrazinolysis peak spectra at 42.6 min in CHY-TPCK

digest corresponding to Histidine – Adduct……………………………...88

Figure 2-11. LC MS Ion Trap Separation of hydrazine digests of CHY-TPCK (top) and

CHY (bottom)……………………………………………………………91

Figure 2-12. LC MS Ion Trap hydrazinolysis peak spectra at 46.4 min in CHY-TPCK

digest corresponding to Histidine – Adduct……………….…………….91

CHAPTER III

Figure 3-1. Sampling sites in Beira Lake (A) and Bolgoda Lake (B)………………109

Figure 3-2. Relative distributions of PAHs for four sites in the water column and

sediments in Beira Lake………………………………………………...112

xv Figure 3-3. Relative distributions of PAHs for six sites in the water column and

sediments in Bolgoda Lake……………………………………………..115

Figure 3-4. Correlation between EC50 values obtained by Microtox assays and total

PAHs for the sediments………………………………………………...120

APPENDIX A

Figure A-1. Tandem mass spectra of peptide from a CC-NBS-LRR RGA type protein

(Helianthus annuus) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...126

Figure A-2. Tandem mass spectra of polypeptides from Lectin (Helianthus tuberosus)

as indicated (bold) in the primary amino acid sequence………………..127

Figure A-3. Tandem mass spectra of peptides from Actin (Mesotigma viride) as

indicated (bold) in the primary amino acid sequence in comparison to

primary amino acid sequence of (H. annuus)…………………………..130

Figure A-4. Tandem mass spectra of peptides from Glyceraldehyde 3-phosphate

dehydrogenase B subunit (Arabidopsis thaliana) as indicated (bold) in the

primary amino acid sequence in comparison to primary amino acid

sequence of Glyceraldehyde 3-phosphate dehydrogenase (H. annuus)...133

Figure A-5. Tandem mass spectra of peptide from Glycolate Oxidase (Arabidopsis

thaliana) as indicated (bold) in the primary amino acid sequence……..135

Figure A-6. Tandem mass spectra of polypeptide from Chitinase (Helianthus annuus)

as indicated (bold) in the primary amino acid sequence………………..137

xvi Figure A-7. Tandem mass spectra of peptide from 33 kDa Oxygen-evolving protein

(Arabidopsis thaliana) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...138

Figure A-8. Tandem mass spectra of peptide from photosystem II protein D1

(Helianthus annuus) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...139

Figure A-9. Tandem mass spectra of peptide from Triosephosphate Isomerase (TIM)

(Arabidopsis thaliana) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...140

Figure A-10. Tandem mass spectra of hevein-like antimicrobial (Euonymus europaeus)

as indicated (bold) in the primary amino acid sequence………………..142

Figure A-11. Tandem mass spectra of ATP synthase SF1 alpha subunit (H. annuus) as

indicated (bold) in the primary amino acid sequence…………………..143

Figure A-12. Tandem mass spectra of Drought-induced protein SDi-6 (H. annuus) as

indicated (bold) in the primary amino acid sequence…………………..145

Figure A-13. Tandem mass spectra of FMTEVR fragment matched to

Glucosyltransferase-like protein (Arabidopsis thaliana) as indicated (bold)

in the primary amino acid sequence…………………………………….146

Figure A-14. Tandem mass spectra of Quinone oxido-reductase-like protein (Helianthus

annuus) as indicated (bold) in the primary amino acid sequence………148

Figure A-15. Tandem mass spectra of Phenylcoumaran benzylic ether reductase

homolog (Tsuga heterophylla) as indicated (bold) in the primary amino

acid sequence…………………………………………………………...149

xvii Figure A-16. Tandem mass spectra of peptide from Triosephosphate Isomerase (TIM)

(Arabidopsis thaliana) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...152

Figure A-17. Tandem mass spectra of peptide from Triosephosphate Isomerase

(Arabidopsis thaliana) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...154

Figure A-18. Tandem mass spectra of peptide from Oxygen-evolving enhancer protein

1, chloroplast precursor (OEE1) (Arabidopsis thaliana) as indicated (bold)

in the primary amino acid sequence…………………………………….157

Figure A-19. Tandem mass spectra of polypeptide from Chitinase (Helianthus annuus)

as indicated (bold) in the primary amino acid sequence………………..159

Figure A-20. Tandem mass spectra of polypeptide from HSP70-related protein (H.

annuus) and HSP70 (Nicotiana benthamiana) as indicated (bold) in the

primary amino acid sequence………………………………...…………162

Figure A-21. Tandem mass spectra of polypeptide from cytosolic phosphoglycerate

kinase (H. annuus) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...164

Figure A-22. Tandem mass spectra of polypeptides from cytochrome f (H. annuus) as

indicated (bold) in the primary amino acid sequence…………………..165

Figure A-23. Tandem mass spectra of polypeptides from malate dehydrogenase

precursor (Medicago sativa) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...168

xviii Figure A-24. Tandem mass spectra of polypeptides from chloroplast ferredoxin-NADP+

oxidoreductase precursor (Capsicum annuum) as indicated (bold) in the

primary amino acid sequence…………………………………………...170

Figure A-25. Tandem mass spectra of polypeptides from NADPH oxidoreductase,

putative (Arabidopsis) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...173

Figure A-26. Tandem mass spectra of polypeptides from malate dehydrogenase

precursor (Medicago sativa) as indicated (bold) in the primary amino acid

sequence………………………………………………………………...174

Figure A-27. Tandem mass spectra of polypeptide from Chitinase (Helianthus annuus)

as indicated (bold) in the primary amino acid sequence………………..177

Figure A-28. Tandem mass spectra of polypeptide from villin 3 (Arabidopsis) as

indicated (bold) in the primary amino acid sequence…………………..178

Figure A-29. Tandem mass spectra of peptide from photosystem II protein D1

(Helianthus annuus) as indicated (bold) in the primary amino acid

sequence……………………………………………………………...…180

Figure A-30. Tandem mass spectra of peptides from Cytokinin-O-glucosyltransferase

(Medicago truncatula) as indicated (bold) in the primary amino acid

sequence……………………………………………………………...…182

Figure A-31. Tandem mass spectra of peptides from ABA/WDS stress induced (Orytza

sativa) as indicated (bold) in the primary amino acid sequence………..184

Figure A-32. Tandem mass spectra of peptides from NAC Domain protein (Orytza

sativa) as indicated (bold) in the primary amino acid sequence………..186

xix Figure A-33. Tandem mass spectra of peptides from a similar to 1-aminocyclopropane-

1-carboxylic acid oxidase (Arabidopsis) as indicated (bold) in the primary

amino acid sequence……………………………………………………187

Figure A-34. Tandem mass spectra of peptide from 1-aminocyclopropane-1-carboxylic

acid oxidase (Rosa hybrid cultivar) as indicated (bold) in the primary

amino acid sequence……………………………………………………189

Figure A-35. Tandem mass spectra of polypeptide from Jacalin-like protein LEM2

(Hordeum vulgare) as indicated (bold) in the primary amino acid

sequence………………………………………………………………..190

Figure A-36. Tandem mass spectra of polypeptide from hevein-like antimicrobial

protein (Euonymus europaeus) as indicated (bold) in the primary amino

acid sequence…………………………………………………………...191

Figure A-37. Tandem mass spectra of polypeptide from HSP 70 protein (Arabidopsis)

as indicated (bold) in the primary amino acid sequence………………..192

Figure A-38. Tandem mass spectra of polypeptide from metal ion binding protein

(Arabidopsis) as indicated (bold) in the primary amino acid sequence...196

Appendix B

Figure B-1. Chromatographic apparatus designed for whole protein separation……198

Figure B-2. Column switch diagram of offline cap-trap enrichment and online sample

introduction……………………………………………………………199

Figure B-3. Chromatographic apparatus designed for small molecule/peptide

separation with post column split………………………………………200

xx Figure B-4. UV spectrum analysis of CHY and CHY-TPCK samples enzymatic

activity by reaction with BTEE observed at 256 nm…………………...201

xxi

CHAPTER I

LC ESI ION TRAP MS DETERMINATION OF ALTERED

PROTEIN EXPRESSION IN HELIANTHUS ANNUUS

AFTER EXPOSURE TO HEAVY METALS As, Cd, Cr, Ni

AND Pb

1.1. Introduction

Within the diverse realm of Bioanalytical Chemistry one of the most recently

blossoming fields of research is Proteomics, the study of proteins, their structure,

function, alterations, expression and their general role in biochemistry. The recent

explosion of research ventures in the field of Proteomics over the last few decades is due

largely to the evolution of modern technology that has expanded the range of focus and

depth of information that can be obtained from a biological sample. The researcher now has access to a vast resource of documented and developing techniques for studying,

manipulating and directing a biological sample’s molecular environment, interactions, structure, and expression profiles. Extensive databases of knowledge are available for each method described and the results attained by altering gene sequences and protein

expression. Coupled to all this is the exponentially expanding field of Bioinformatics,

online databases that contain a comprehensive listing of nearly all published or expressed

protein structures and DNA sequences.

Proteomics was defined in 1838 by Berzelius when he put forth the name “protein”

(from the Greek proteios, “primary”) for the complex organic nitrogen-rich substance

found in the cells of all living organisms. Over the course of the nineteenth century

researchers identified many of the 20 common amino acids that make up proteins. In

1864 the first protein, hemoglobin, was crystallized by Hoppe-Seyler. In 1894 a lock-

and-key analogy for enzyme-substrate interactions was proposed by Fischer. Three years

later, in 1897, Buchner and Buchner created enzymology by demonstrating that cell-free

extracts of yeast ferment sucrose to form carbon dioxide and ethanol. In 1926 Sumner

crystallized urease in pure form, demonstrating that proteins could possess the catalytic

activity of enzymes. At the same time Svedberg developed the first analytical ultracentrifuge and used it to estimate the correct molecular weight of hemoglobin. In

1933 Tiselius introduced electrophoresis for separating proteins in solution. One year later, in 1934 Bernal and Crowfoot presented the first detailed x-ray diffraction patterns of a protein, obtained from crystals of the enzyme pepsin. In 1942 Martin and Synge developed chromatography, a technique now widely used to separate proteins. In 1951

Pauling and Corey proposed the structure of a helical conformation of a chain of L-amino acids, the α helix, and the structure of the β sheet, both of which were later found in

many proteins. In 1955 Sanger completed the first analysis of the amino acid sequence of

a protein, insulin. In 1956 Ingram produced the first protein fingerprints, showing that

the difference between sickle-cell hemoglobin and normal hemoglobin is due to a change

in a single amino acid. In 1960 Kendrew described the first detailed structure of a protein

(sperm whale myoglobin) to a resolution of 0.2 nm, and Perutz proposed a lower-

resolution structure for hemoglobin. In 1963 Monod, Jacob, and Changeux recognized

that many enzymes are regulated through allosteric changes in their conformation. This

body of initial experimental work set the foundation for a wide range of scientific

endeavors in the realm of Proteomics, limited only by the inventiveness of the researchers

and the availability of precision instrumentation.

Since the 1960’s a vast amount of research has been performed on proteins.

Extensive data on the sequences, functions, expression patterns, and folded structures of

proteins have been analyzed, documented and entered into globally accessible online

databases. The largest hurdle the biochemical researcher has had to overcome has been

to develop the means to more thoroughly analyze the complex biological sample. The

early experiments described above involved incredibly lengthy and time-consuming

isolation and purification steps. Sample size had to be very large since instrument

sensitivity was very poor. As Proteomics developed as a field Chromatography and

Electrophoresis came to the forefront of useful analytical methodologies for the

separation of the complex sample for further analysis. These techniques allow mixtures

of proteins and peptides to be separated into their individual components according to

their inherent characteristics; size, iso-electric point, electronic interaction, hydrophobicity, and relative pH.

The 1970’s saw the development of many reliable chromatographic methods for the biochemical researcher. Pressure liquid chromatography was designed to decrease flow through time and reduce the purification times for compounds being isolated. High- pressure liquid chromatography (HPLC) was the next development, which led to the development of on-line detectors and new and improved column packing materials.

Amongst these new packing materials were hydrophobic carbon chain based molecules, termed reverse phase liquid chromatography, these facilitated improved separation between very similar biological compounds. Continued method development led to improvements in separation, identification, purification and quantification far above the previous techniques. The incorporation of computers and automation in the 1980’s added to the convenience and reproducibility of HPLC.

Several branches of HPLC have been developed that take advantage of specific properties by which biomolecules can be separated. Ion exchange chromatography separates molecules based on how they interact with charged groups immobilized on the

column matrix. The binding affinity of a given peptide is a result of its net charge, determined by the pH of the solution, and the presence of other ions in the sample that can compete for binding with the ion exchanger. A strong ion exchange column is ideal for peptide separation due to the wide variety of charged residues and the ability to change their charge potential by altering the pH of the mobile phase [1].

Gel filtration chromatography or size exclusion chromatography utilizes a column packing consisting of various gel beads created with pores manufactured to a preset size.

As larger molecules pass around these beads they elute from the column faster than smaller molecules that are capable of interacting or passing thru the pores [1].

Affinity chromatography is designed to utilize the ability of proteins to specifically bind to a molecule or ligand in a non-covalent fashion. The column packing matrix is created to contain a protein specific ligand so that when a complex mixture is passed through the chromatographic material, the protein of interest binds to the packing and all other molecules are eluted. In this technique the isolated protein can then be eluted by changing the buffer conditions to release it from the matrix [1].

Of primary interest to our research group is reversed phase chromatography, the separation of biomolecules based upon their hydrophobic interactions with a non-polar packing material. Functional groups interact or associate with the hydrophobic groups on the column backbone. Separation is accomplished by changing the mobile phases over a gradient to vary the relative polarity. [1]

Once the biochemical researcher has separated the complex mixture then he/she has to

develop a method to identify the analytes obtained. Initially this identification step was

also very time intensive, it involved obtaining structural information from a variety of

spectroscopic analyses and performing chemical reactions to determine the sequence

information of the protein. The need for a more thorough analyte identification methodology was addressed in two distinct ways. The first involved the development of

X-ray crystallography in the 1930s and nuclear magnetic resonance (NMR) spectroscopy in 1945. These methods allowed a new means of determining analyte structure and sequence but carried some limitations as both methods required high purity, relatively large sample size and the ability of the molecule to form a crystal. The development of computer technology and advanced algorithm programming provided techniques which today are extremely fundamental in understanding molecular conformation and structure, but they are an “offline” step that still depends on the somewhat difficult step of the creation of effective crystals. The second method developed for analyte identification also premiered in the 1930s, and was coined the “Mass Spectrometer”.

The first clear demonstrations of mass spectrometry came at the end of World War I by Francis W. Aston and Arthur J. Dempster, in Cambridge and Chicago, respectively.

Dempster used a magnetic analyzer that focused ions into an electrical collector, while

Aston used both electrostatic and magnetic fields to focus ions on a photographic plate.

Their work continued, along with that of Joseph H. E. Mattauch, R. F. K. Herzog,

Kenneth T. Bainbridge, and Alfred O. C. Nier. Nier then incorporated developments in vacuum technologies and electronics for power supplies and ion detection, significantly improving magnetic focusing instruments. Through this technique he established that it

was not necessary to send ions along their entire path of a semicircle within an analyzer completely confined between the poles of a massive magnet (prior standard practice).

Instead good results could be obtained by sending ions through a comparatively modest wedge-shaped sector magnetic field. More practical electron-bombardment ion sources, along with several other crucial aspects of construction and technique, also brought improvements in performance, convenience, and costs. Double-focusing machines were also greatly refined, attaining greater precision by adding an electrostatic analyzer.

Analyzers based on the different times of flight over a set path for different accelerated ions, and others that used various types and combinations of fields also had some success.

In 1953 Wolfgang Paul and his colleagues initiated development of the quadrupole mass analyzer (and ion trap), using crossed radio frequency and electrostatic fields. The middle and later 1950s saw the first wave of efforts to couple the mass spectrometer with other significant types of instruments, creating, in this case various types of gas chromatograph mass spectrometers. In the 1960s, chemical ionization mass spectrometry

(or CIMS) and field desorption MS (FDMS) emerged. In the 1970s secondary ionization

MS, Fourier transform MS, plasma desorption MS, electrohydrodynamic MS, laser desorption MS, thermal desorption MS, spark source MS, and glow discharge MS were invented or developed significantly. The 1980s saw the development of resonance ionization MS, matrix-assisted laser desorption MS, fast atom bombardment MS and its continuous flow transformation. Following this was the development of ion trap MS, a dramatic advance in electrospray MS, and the subsequent development of modern liquid chromatography MS.

Our current applications of two dimensional liquid chromatography coupled with mass spectrometry involves the complete separation and subsequent identification of

complex mixtures of biomolecules. In theory, samples containing 1000 or more proteins

can be digested and still be resolved to complete separation and identification, limited

only by the specificity of the equipment, the skills of the researcher and the extensiveness

of the bioinformatics library. Virtually any sample can be analyzed with the diverse

array of chromatographic techniques and mass spectrometers available to the researcher.

The versatility of this method stems from coupling multiple stages of chromatographic

separation to isolate many smaller fractions, from the original complex mixture, that can

be more thoroughly analyzed by the multiple MS. A researcher has available the entire host of available column / gradient separation techniques of liquid chromatography. This methodology is most often coupled with digestion of the protein sample, an enzymatic process of reducing the protein into many smaller peptide fragments. These peptide fragments can then be identified using the mass spectrometer and characterized completely with tandem MS evaluations.

Protein digestion is a common technique used to evaluate the amino acid composition and to determine structural information of proteins. Information for hundreds of thousands of proteins exist within the bio-informatics databases, including information on predicted peptide cleavage patterns from varying enzymatic digests. Among the most common enzymes utilized are the Serine Peptidases, a class of digestive enzymes whose active site is composed of a catalytic triad, a unique arrangement of an aspartic acid, a histidine and a serine residue that are highly selective to specific peptide bonds due to the proteins folded structure. Located next to this catalytic triad is a hydrophobic pocket of

varying characteristics for each enzyme that alters the specificity of the cleavage patterns.

Trypsin is an enzyme specific for cleaving the peptide bond on the carbonyl side of the

basic residues arginine and lysine. Chymotrypsin is an enzyme specific for cleaving the

peptide bond on the carbonyl side of the bulky aromatic side chains phenylalanine,

tryptophan and tyrosine. Elastase is an enzyme specific for small neutral residues,

particularly alanine. These enzymes used sequentially for multiple digest and sequence

determination experiments on a sample can allow for detailed secondary protein structure

to be determined.

The post-digest peptide fragments obtained couple excellently with reverse phase

liquid chromatography. The peptide fragments possess all the properties of their

constituent amino acid residue side chains in addition to their c and n-termini. The

protein digest is enriched and loaded on a hydrophobic captrap, to concentrate and

remove buffer contaminants by reverse phase microcolumns in order to increase signal- noise ratios and sensitivity before MS analysis. This does allow for the loss of small and hydrophyllic peptides but more importantly will wash the sample of any residues or compounds that can be damaging or interfere with the sensitive ion trap. After washing

in the trap the peptides are then eluted and passed through a C-18 reversed phase column allowing separation by hydrophobic interaction. The end result is a potential reduction in the number of peptides passing through the mass spectrometer by some three orders of magnitude from that of the original complex. The coupling of the Reversed Phase chromatography step with the mass spectrometer requires a means of ionizing the peptide fragments. The mobile phase from the chromatography step is most commonly a mixture

of water and acetonitrile or water and methanol, both of which couple excellently with

the electro-spray ionization technique.

Electro-Spray Ionization (ESI) is a technique that facilitates the transfer of ions from

the liquid phase to the gas phase. In ESI, charged droplets are produced from electrolytes

dissolved in the solvent and nebulized by use of a gas stream upon entry to the MS. The

charged droplets are then dried and reduced in size by solvent evaporation and repeated

splitting, eventually creating very small highly charged droplets capable of producing gas

phase ions. This ionization technique allows the formation of analyte ions in the gas

phase and analysis of the charged molecules by their interaction with the mass

spectrometers detector [2]. An Ion Trap MS can allow for multiple tandem MS analysis,

currently up to 12+ times daughter analysis of a single biomolecule. 2-D LC separation

with MSn ion trap analysis can in theory allow tens of thousands of proteins to be simultaneously analyzed, and this couples with high throughput, an excellent dynamic range (able to analyze 106:1 relative protein abundance) and a very flexible methodology

with variance available in LC conditions and columns, as well as a variety of enzymatic

digests available.

Within the field of Proteomics extensive work has been undertaken using the

technique of multidimensional liquid chromatography coupled mass spectrometry (LC-

MS). LC-MS techniques have been combined with 1 dimensional and 2 dimensional

electrophoretic separation for direct and systematic identification of expressed proteins

from prepared biological media. Proteins resolved by 2D gel electrophoresis can be

enzymatically digested in–gel, or digested during blotting onto membranes containing

immobilized trypsin. Trypsin cleaves after the basic residues arginine and lysine, if not

followed by proline along the polypeptide chain. Each of the tryptically produced peptides will therefore have a basic arginine or lysine c-terminus residue in addition to its n-terminus, both of which are capable of retaining a positive charge. This digest method couples perfectly with ESI ion trap analysis since each peptide created can retain at minimum a 2+ charged state allowing for in depth fragment detection in MS/MS ion trap analysis providing in depth structural definition.

Phytoremediation, the use of plants to restore soil and water contaminated with pollutants, is considered a promising approach due to its low cost and the minimal impact the procedure has on the environment. Interest in the use of plants for environmental restoration has grown following the discovery that some plants are capable of accumulating toxic metals at levels many times greater than common nonaccumulator plants. These plants are described as hyperaccumulators or hyperacumulating plants.

There are over 400 plant species that are considered metal hyperaccumulators of one or more toxic metals [3]. Helianthus annuus (Dwarf Sunflower) is capable of hyperaccumulating several metals including Ni, Cd, Cu, and Cr [4]. As metal accumulation is of serious interest and since accumulation of metals is toxic to plants, hyperaccumulating plants must have developed mechanisms of tolerating metal accumulation. Mechanisms underlying hyperaccumulation of toxic metals are complex.

One major mechanism might be to counteract the oxidative stress initiated by heavy metals by induction of antioxidative enzymes [5]. Another defense mechanism appears to involve induction of metal binding proteins, e.g., phytochelatins (PC) and metallothioneins (MT) [6].

These small sulfur-rich peptides have been the focus of recent studies. For instance, he

synthesis of PC has been shown to be induced by a variety of different metals [7,8]

Rendering the plants tolerant to accumulation of such metals as copper and cadmium

[9,10]. On the other hand, some proteins that are expressed in response to the metal

stress are not considered as metal chelators. Notable examples are chitinases and heat

shock proteins [11,12], although the significance of their expression in the metal

hyperaccumulation is not understood.

Proteomics has recently been applied to study the regulation of proteins as related to

metal stress in plant species. To date few studies have observed the specific protein

expression in H. annuus or any hyperaccumulator plants to specific metal stress [11,13].

The purpose of the present study is to identify and characterize proteins that are observed

in increased spot intensity in response to four metals (Cd, Cr, Ni, and Pb) that

hyperaccumulate in H. annuus. The proteins may not play a direct role in the transport or

sequestration of toxic metals but could be involved in mediating metal tolerance in other

ways. Recent studies of metal accumulation have induced oxidative stress responses in

plants [14] in addition to a variety of other observed proteins [15-18]. These responses leave open the question of what possible response stress mechanisms are being initiated on metal accumulation. Many of the observed proteins in these studies are not considered to be metal binding, although their biological function may in some way

contribute to the ability of the plants to deal with metal stress. Many of these studies

leave unresolved questions as to the detailed proteomic effects of metal exposure [17,18].

This study aims to further expand some of the proteomic findings of the exposure to

heavy metals on a known hyper-accumulator species, H. annuus.

1.2. Methodology

1.2.1. Plant Growth and Exposure

Sunflowers were exposed to heavy metals in solutions containing single metals (As,

Cd, Cr, Ni or Pb) and combinations of Cd, Cr and Ni, with or without As or Pb. Each

heavy metal was added at 30 mg/L. The 30 mg/L refers to the concentration of the

individual metal, not the compound added. The metals were applied as As5-,

. 2+ . 3+ . 2+ . (Na2HAsO4 7H2O), Cd (CdSO4 8H2O), Cr (CrCl3 6H2O), Ni (NiSO4 6H2O), and

2+ Pb (Pb(NO3)2.

Experiments were conducted with two different Helianthus annuus cultivars

(Sundance and Teddy Bear). Seeds of each cultivar were grown hydroponically in

Rockwool, a non-reactive, nonabsorbent support system and sustained with a standard

hydroponic nutrient solution (Cropking Corp, Seville, OH). The seeds were grown in a

greenhouse illuminated with natural light. The average temperature of the greenhouse

was 28oC (winter) and 35oC (summer) in the day and 20oC at night. After a four week growth period, seven sunflowers were transferred to a PVC trough. One set of plants was then subjected to a 4 L solution containing 30 mg/L of the toxins as described above.

Solutions were introduced at a complete recycle rate of 6 gallons per hour. After 17 days of exposure, the plants were harvested and the leaves were then sectioned and weighed.

The tissues were used for analysis of metal levels, and for protein and RNA isolation.

Plant growth and exposure was conducted by Dr. Teresa Cutright at the University of

Akron, Department of Civil Engineering [11].

1.2.2. Protein Extraction and Gel Separation

The plant tissues were mashed with mortar and pestle in liquid nitrogen, and 100-200

mg and were placed in extraction buffer. The plant extraction solution FocusTM Plant

Proteome (G-Bioscience, St. Louis, MO) was used following the manufacturer’s

instructions, then centrifuged for 20 min at 15,000 x g. The supernatants were collected

and the protein concentrations were measured using the Bradford method with albumin as

the reference protein solution. Sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) was performed to establish distribution of proteins,

molecular mass of the proteins present in the control and the metal-exposed plant

biomass. Later experiments first separated proteins by isoelectric focusing prior to

electrophoretic separation. Gels were stained with Coomassie Brilliant Blue and protein

bands (1 dimensional) or spots (2 dimensional) of interest gels were excised for further

digestion and analysis. Protein extraction and gel separation was performed in Dr Harry

vanKeulen’s laboratory by Chamari Hasintha, Cleveland State University [11].

1.2.3. Sample Preparation and Tryptic Digest

Trypsin was obtained from Promega or Pierce. Protein digestion was performed on

gel bands (1-D) and gel spots (2-D) of interest that were excised, cut into approximately

1mm3 pieces and stored at -20° C until digestion. In preparation for digestion gel pieces

were washed/destained with 200 µL wash reagent (50% Et-OH / 5% acetic acid / 45%

H2O), for 1 h. The wash was discarded and the wash step repeated for an additional

hour. Samples were then reduced by adding 30 µL 100 mM dithiothreitol and incubating

the sample at 60° C for 10 min, reducing buffer was then discarded. Samples were then alkylated by adding 30 µL 100 mM iodoacetamide and incubating in the dark at room temperature for 1 h, alkylation buffer was then discarded. Gel pieces were washed with

200 µL wash reagent for 1 h. The wash was discarded and the wash step repeated for an additional hour. Gels were then dehydrated by adding 200 µL acetonitrile for 10 min.

The acetonitrile was discarded and samples were rehydrated in 200 µL 100 mM ammonium bicarbonate for 5 min. Then the ammonium bicarbonate was discarded and samples were again dehydrated with 200 µL acetonitrile for 10 min. Next, the acetonitrile was discarded and the samples were dried in SpeedVac for approximately 3-5 min. Finally, samples were rehydrated in 50 µL trypsin (20 ng/µL in 50 mM ammonium bicarbonate) for 10 min on ice. Excess Trypsin solution was then removed and 5 µL of

50 mM ammonium was added to submerge the gel pieces. Samples were incubated at

37° C for 4 h or alternatively held at room temperature (23-25° C) overnight.

After the digestion step was completed, samples were microcentrifuged and the supernatant digest mixture collected. 30 µL extraction reagent (50% ACN / 5% formic acid / 45% water) was added to the remaining gel pieces. The sample was briefly vortexed and left for 10 min. The extraction mixture was removed and added to the digest mixture, the extraction step was repeated once. This additional extraction step can slightly increase digest yield and also serves to terminate any continuing trypsin activity.

Combined supernatants were placed in a SpeedVac to reduce volume to approximately 10

µL, about 45 – 60 min. Sample volumes were reconstituted to approximately 25 µL total volume with 1% acetic acid. Samples were now ready for LC-MS analysis.

HPLC ESI-Ion Trap MS was carried out by injecting the sample offline into a secondary LC loop using a loading phase of 2% ACN in H2O with 0.2 % HCOOH, the molecules were enriched on a C-18 CapTrap (MicromResources). The sample was washed for 10 minutes in the CapTrap and then the main column switch brought the

CapTrap online into the primary Agilent LC mobile phase ramp as described below. Ion

Trap conditions were set initially to those determined for small molecule analysis and optimized as needed.

1.2.4. LC Separation

HPLC analysis was carried out on an Agilent 1100 series pump (Agilent, Palo Alto,

CA). Samples were loaded offline in 10 µl aliquots onto a Peptide C-18 CAPTRAP

(Michrom Bioresources, Auburn, CA) for enrichment and washing and were subsequently separated on a reverse-phase C-18 MS column (100 x 0.3 mm, 5 µm, 300

Å; Vydac, Hesperia, CA). Initial Peptide Cap Trap loading was performed using the following conditions: 10 µl/min using a 2% acetonitrile (ACN), 1% formic acid solvent mixture in water for 10 min on a LV-10 ADVP pump (Shimadzu, Colombia, MD). At 10 min loading solvent flow switched to primary LC flow. Peptides were resolved using the following conditions: flow rate of 5 µl/min, linear gradient from 5 - 40% buffer B (0.1% formic acid, 99.9 % ACN) in solvent A (0.1 % ACN, 0.1 % formic acid in water) for 70 min, then 40 - 80% B for 30 min.

1.2.5. MS Analysis

The samples were analyzed using an EsquireHCT Brucker Daltonics mass spectrometer equipped with a standard Agilent electrospray (ESI) ion source (Brucker

Daltonics, Billerica, MA). The instrument settings were as follows: nebulizer-11.0 psi,

flow rate of dry gas 5.0 Liter/min; temperature 310oC; ESI voltage 3.0 kV; scan mode-

standard-enhanced with range 50 - 3000 m/z, speed 8, 100 m/z/s; trap settings-positive polarity, ICC Smart Target-105; maximum accumulation time 2 x 104 ms, scan-350-1700 m/z; and target mass-800 m/z. MS/MS data were acquired using an Auto MSn feature that functions in the “Triple Play’ type data-dependent mode, a total scan that included three scan events: a full range MS scan; a narrow-range, high resolution zoom scan on a selected ion from the MS scan; and a MS/MS scan of the selected ion.

1.2.6. MASCOT Bioinformatics

The data were searched against NCBI using the MASCOT soft ware. Additional

BLAST search algorithms were exhaustively performed against the NCBI database using individual peptide strings and whole protein sequences for potential matches.

Two sets of Mascot MS/MS ion search criteria were applied to both the Veridiplantae and the Arabidopsis thaliana taxonomy filters. The initial search parameters for trypsin digestion allowing one miscleavage, variable modification—oxidation of methionine,

MS/MS tolerance of 0.5 Da, and an ion score cut-off of 20. A second intentionally broad search was applied, allowing a wider range of possible peptide matches from many species to compliment the somewhat sparse H. annuus data base, results from this search are excluded from the tables.

1.3. Results

1.3.1. Expression of proteins in Sundance

1.3.1.1. Expression of proteins in response to arsenic and other metals in Sundance

Hydroponic Sundance root samples 1-D PAGE analysis revealed three distinct spots around markers at 30 and 66 kDa (Fig. 1-1). The mass spectra of tryptic digests of these spots indicated a positive match to two proteins in the first spot, and one protein in each

of the other spots (Table 1-I). The first peptide from the first spot matched the peptide

YGGMVMoIWDR, Mass 1095.52 Da (549.27, 2+) to Chitinase H. annuus a 32 kDa

protein (Fig. 1-2). MS/MS fragmentation yielded twelve fragment ions of the nineteen

most intense peaks matching the expected masses of b2, b3, b4, b6, b7, y1, y3, y4, y5, y6, y7, y8. The second peptide from the first spot matched the peptide

LENGQSWQITVAPGTAQAR, mass 2026.02 Da (1014.05, 2+), (676.37, 3+) to an

unknown protein sharing close homology to pathogenesis-related protein 5-1 H. annuus a

25 kDa protein (Fig. 1-3). MS/MS fragmentation of the 2+ charged peptide yielded

thirteen fragment ions of the fourteen most intense peaks matching the expected masses

0 * 0 ++ of b11, b11 , b12, b18 , y7, y8, y9, y10, y11, y12, y12 , y13 , y14. MS/MS fragmentation of

the 3+ charged peptide yielded forty-one fragment ions of the forty-four most intense

* 0 0 * *++ 0++ peaks matching the expected masses of b5, b6, b6 , b6 , b8 , b9, b10 , b10 , b10 , b11,

++ *++ 0 0++ ++ *++ 0++ *++ ++ ++ *++ 0++ b11 , b11 , b11 , b11 , b12, b12 , b11 , b12 , b13 , b15 , b16 , b16 , b16 ,

++ * ++ 0 * 0 ++ ++ ++ *++ 0++ *++ b18 , y2, y6, y7, y7 , y8, y8 , y8 , y9, y9 , y9 , y11 , y13 , y14 , y16 , y16 , y17 ,

0++ y17 . The second spot, below the 66 kDa marker, matched the peptide DEKPEGTSR, mass 1016.53 Da (340.06, 3+) to CC-NBS-LRR RGA protein, H. annuus, a 19 kDa

protein (Fig. A-1). MS/MS fragmentation yielded seven fragment ions of the nine most

0 intense peaks matching the expected masses of b4*, b4 , y3++, y4++, y5++, y6++, y8++.

The third spot, around the 66 kDa marker, matched multiple peptides VVGFFGR, mass

780.43 (391.21, 2+), QTYGPYGTNGGTDFSCPIAK, mass 2075.93 (692.93, 3+),

NGSLSTIDIADDEEIIEINGK , mass 2245.10 (1123.59, 2+) corresponding to a 15 kDa subunit of Lectin Helianthus tuberosus (Fig. A-2). MS/MS fragmentation of VVGFFGR yielded five fragment ions of the six most intense peaks matching expected masses of b2,

y3, y4, y5, y6. MS/MS fragmentation of QTYGPYGTNGGTDFSCPIAK yielded

seventeen fragment ions of the thirty-five most intense peaks matching expected masses

++ * ++ ++ *++ 0++ *++ 0++ ++ ++ ++ of b4 , b9 , b10 , b13 , b15 , b15 , b18 , b18 , b19 , y6, y7, y8, y10 , y11, y13 ,

++ 0++ y15 , y19 . MS/MS fragmentation of NGSLSTIDIADDEEIIEINGK yielded twenty- six fragment ions of the twenty-two most intense peaks matching expected masses of b6,

0 0 0 ++ b6 , b7, b9, b10 , b15, b15 , b16, b17, b18, y3, y4, y5, y6, y7, y9, y10, y11, y12, y12 , y13, y14,

0 y14 , y15, y17.

Figure 1-1. 1-D Gel Separation Sundance Hydroponic As [11]

Table 1-I. 1-D Hydroponic Sundance Root As, Cd, Cr, Ni

Marker Region Theoretical Protein Peptide kDa Mass kDa Accession no. sp. Name Score (Coverage) Peptides

30 32 gi|126723930 H. annuus Chitinase 72 1 (2%) YGGVMoLWDR

Unnamed 30 25 gi|13938981 H. annuus protein 64 1(9%) LENGQSWQITVAPGTAQAR

CC-NBS- LRR 60 19 gi|49425431 H. annuus RGA 31 1 (3%) DEKPKEGTSR

VVGFFGR, H. QTYGPYGTNGGTDFSCPIAK, 66 15* gi|11559262 tuberosus Lectin 211 3 ( 18%) NGSLSTIDIADDEEIIEINGK

(A)

MEFTHPALLLLLFITVFSFLKPSTAAGIATYWGQQSDDTEGTLEAACATGNYQFV

NIAFLSTFGNNQQPVLNLAAHCDPASTCSRYSSQIKACQAQNVKVFLSIGGQRGS

YSLSSPQDAQQVADFLWNTYLGGQPATRPLGDAVLDGIDFDIEQGTDQFWSDLA

KAPAAYSSQKKVYLSAAPQCPFPSGDVRNQLLPAIREGLFDYVWVQFYNNEQCQ

YGANADALLARWNEWTQVTTNTIFLGLPAAASGAAPSGGYIPPDILTSQILPSIKS

SPKYGGVMLWDRFYDKQSGYSDAIKGSIN

(B)

Figure 1-2. Tandem mass spectra of peptide (A) from Chitinase (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

(A)

(B)

MTCAKNLLLSITLLSIGCFTLTRGATFDVINQCQYPVWAAWASTTPGGGKRLEN

GQSWQITVAPGTAQARIWGRTGCNFDANGRGRCDTGDCNGMLECQGYGAPP

NTLAEFALNQDNNNDFVDISLVDGFNIPMEFSPVGASCKTMRCAGNLNGECPNE

LRTQGGCNNPCTVYKTNEYCCTNGPGSCGPTPLSRFFKDRCPDAYSYPQDDPTSL

FTCPGGTNYKVVFCP

(C)

Figure 1-3. Tandem mass spectra of +2 (A) and +3 (B) peptides from an unknown

protein type with high sequence homology to pathogenesis-related protein 5-1 (H. annuus) as indicated (bold) in the primary amino acid sequence (C).

1.3.1.2. Expression of proteins in response to arsenic only in Sundance

Hydroponic Sundance leaf samples 1-D PAGE analysis revealed one very distinct spot around the marker region at 45 kDa. The mass spectra of the tryptic digest of this spot indicated positive matches to three proteins (Table A-I). The first set of peptides,

HQGVMVGMGQK mass 1170.56 (391.22, 3+), AVFPSIVGRPR mass 1197.70 (400.28,

3+), SYELPDGQVITIGNER mass 1789.88 (895.93, 2+), VAPEEHPILLTEAPLNPK

mass 1967.07 (656.81, 3+), match to Actin, Mesostigma viride, a 42 kDa protein with

high homology to Actin, H. annuus (Fig. A-3). MS/MS fragmentation of

HQGVMVGMGQK yielded five fragment ions of the fifteen most intense peaks

++ ++ ++ ++ *++ matching the expected masses of y3 , y4 , y5 , y7 , y7 . MS/MS fragmentation of

AVFPSIVGRPR yielded four fragment ions of the five most intense peaks matching the

++ ++ expected masses of b2, b3, y8 , y9 . MS/MS fragmentation of SYELPDGQVITIGNER

yielded twenty-six fragment ions of the thirty-four most intense peaks matching the

0 0 0 0 *++ 0 0++ expected masses of b2, b3, b4, b4 , b8 , b9, b9 , b10, b10 , b12, b12 , b12 , b12 , y4, y5,

++ 0 ++ ++ y6, y7, y9, y10, y11, y12, y12 , y12 , y13, y13 , y14 . MS/MS fragmentation of

VAPEEHPILLTEAPLNPK yielded seventeen fragment ions of the thirty-one most

0 ++ ++ ++ ++ intense peaks matching the expected masses of b8 , b9, b10, b10 , b11 , b12 , b13 ,

*++ * ++ b16 , y3, y5, y5 y6, y7, y8, y9, y12, y16 . The second set of peptides, HIQAGASK

mass 810.43 (271.35, 3+), AVSLVLPQLK mass 1066.67 (534.35, 2+), match to

glyceraldehyde 3-phosphate dehydrogenase B subunit, Arabidopsis, a 44 kDa protein

(Fig. A-4). MS/MS fragmentation of HIQAGASK yielded seven fragment ions of the

++ ++ *++ ++ fifteen most intense peaks matching the expected masses of b2, b2 , b4 , b4 , y2 ,

* ++ y2 , y5 . MS/MS fragmentation of AVSLVLPQLK yielded nine fragment ions of the

0 nine most intense peaks matching the expected masses of b2, b4 , b5, b6, y4, y5, y6, y7,

y8. The final peptide, IAIQAGAAGIIVSNHGAR mass 1717.96 (573.81), matched

glycolate oxidase, H. annuus, a 40 kDa protein (Fig. A-5). MS/MS fragmentation of

IAIQAGAAGIIVSNHGAR yielded eighteen fragment ions of the thirty-nine most

* *++ ++ * 0++ ++ intense peaks matching the expected masses of b3, b6 , b6 , b13 , y5 , y7, y7 , y9 ,

++ ++ ++ ++ *++ 0++ ++ *++ ++ y10, y10 , y12 , y13 , y14 , y14 , y14 , y15 , y15 , y16 .

1.3.1.3. Expression of proteins in response to lead and other metals in Sundance

Hydroponic Sundance leaf samples 1-D PAGE analysis revealed two distinct spots,

one around the marker region at 37 kDa and the other just below it. The mass spectra of

tryptic digest of these spots indicated positive matches to two and six proteins

respectively (Table A-II). The first peptide from the first spot, matched the peptide

YGGMVMoIWDR, mass 1095.52 Da (548.75, 2+) to Chitinase H. annuus a 32 kDa

protein (Fig. A-6). MS/MS fragmentation yielded twelve fragment ions of the nineteen

most intense peaks matching the expected masses of b2, b3, b4, b6, y1, y3, y4, y5, y6, y7, y8. The second peptide from the first spot, matched the peptide

GGSTGYDNAVALPAGGR, mass 1561.75 (781.43, 2+), to a 33 kDa oxygen evolving

protein Arabidopsis (Fig. A-7). MS/MS fragmentation yielded twenty-two fragment ions

0 0 of the thirty-eight most intense peaks matching the expected masses of b5, b7, b9 , b10 ,

0 0 ++ ++ * b11, b11 , b12, b12 , b13, b14, b14 , b15 , y4, y5, y5 , y6, y7, y8, y9, y10, y11, y12, y13. The first peptide from the second spot, matched VINTWQDIINR, mass 1313.71 (657.93, 2+), to photosystem II protein D1 H. annuus, a 38 kDa protein (Fig. A-8). MS/MS fragmentation yielded seven fragment ions of the eleven most intense peaks matching the

* expected masses y3, y3 , y4, y5, y6, y8, y10. The second peptides from the second spot,

matched DEFIGKK, mass 835.44 (418.04 2+), VASPQQAQEVHVAVR, mass 1532.81

(511.84, 3+), IIYGGSVNGGNSAELAK, mass 1648.84 (825.33, 2+), to TIM

Triosephosphate Isomerase Arabidopsis, a 33 kDa protein (Fig. A-9). MS/MS fragmentation of DEFIGKK yielded six fragment ions of the twenty most intense peaks

0 * matching the expected masses b1, b3, b3 , b4, y2, y3 . MS/MS fragmentation of

VASPQQAQEVHVAVR yielded twenty-four fragment ions of the thirty-one most

++ 0 ++ ++ ++ intense peaks matching the expected masses b2, b5, b6, b6 , b9 , b10 , y4, y5, y5 , y6 ,

++ ++ ++ ++ *++ ++ ++ ++ ++ *++ 0++ y7, y7 , y8, y8 , y9 , y10 , y10 , y11 , y12 , y13 , y14 , y14 , y14 . MS/MS

fragmentation of IIYGGSVNGGNSAELAK yielded twenty fragment ions of the fifty-six

0 * 0 most intense peaks matching the expected masses b6, b7, b7 , b8, b14, b15, b15 , b15 , b16,

0 ++ * * * ++ *++ 0++ ++ b16 , y3, y8 , y9, y10, y10 , y11, y12, y13, y13 , y14, y14 , y15, y15 , y15 , y15 , y16 .

The third peptide from the second spot, matched GPLQLTWNYNYAQAGR, mass

1850.91 (926.45, 2+), to hevein-like antimicrobial peptide Eunonymus europaeus, a 34

kDa protein (Fig. A-10). MS/MS fragmentation yielded thirteen fragment ions of the

* * fifteen most intense peaks matching the expected masses b4, b6, b7 , b9, b9 , y5, y6, y7,

y8, y9, y10, y11, y12. The fourth peptides from the second spot, matched RPPGR, mass

581.34 (292.09, 2+), LIESPAPGIISR, mass 1251.72 (626.87, 2+), to ATP synthase CF1

alpha subunit H. annuus, a 55 kDa protein (Fig. A-11). MS/MS fragmentation of RPPGR

yielded six fragment ions of the thirty-four most intense peaks matching the expected

* *++ * masses y1, y2, y2 , y2 , y3, y3 . MS/MS fragmentation of LIESPAPGIISR yielded nine

fragment ions of the twenty-nine most intense peaks matching the expected masses y2,

++ 0 y3, y6, y7 , y8, y9, y10, y10 . The fifth peptide from the second spot, matched

SGATGATPPAEAPLVTEEK, mass 1824.91 (913.44, 2+), drought-induced protein SDi-

6 H. annuus a 18 kDa protein (Fig. A-12). MS/MS fragmentation yielded fifteen of the

0 ++ 0++ thirty-five most intense peaks matching the expected masses b7, b7 , b12 , b13, b15 ,

++ * y4, y7, y8, y9, y10, y11, y12, y12 , y13, y17 . The sixth peptide from the second spot,

matched FMTEVR, mass 781.38 (390.90, 2+), glucosyltransferase-like protein

Arabidopsis a 37 kDa protein (Fig. A-13). MS/MS fragmentation yielded four of the five

most intense peaks matching the expected masses b1, b2, b3, b5.

Hydroponic Sundance stem samples 1-D PAGE analysis revealed 1 distinct spots

around the marker region at 39 kDa. The mass spectra of tryptic digest of the spot

indicated positive matches to two proteins (Table A-III). The first peptide matched

SLGADVAIDYTK, mass 1251.63 (626.87, 2+), to Quinone oxido-reductase-like H.

annuus, a 33 kDa protein (Fig. A-14). MS/MS fragmentation yielded twelve of the

0 0 twenty most intense peaks matching the expected masses b2, b6 , b11, b11 , y3, y4, y5,

* y6,y7, y8, y9 , y10. The second set of peptides matched VVILGDGNAK, mass 984.56

(493.31, 2+), DKVVILGDGNAK, mass 1227.68 (614.38, 2+), to phenylcoumaran

benzylic ether reductase homolog TH3 Tsuga heterophylla, a 33 kDa protein (Fig. A-15).

MS/MS fragmentation of VVILGDGNAK yielded nine of the thirteen most intense peaks

* matching the expected masses b2, b3, b4, b6, b6 , b9, y6, y7, y8. MS/MS fragmentation of

DKVVILGDGNAK yielded five of the nineteen most intense peaks matching the

0 expected masses b2 , y6, y7, y9, y10.

Hydroponic Sundance leaf samples (soluble fraction only) 1-D PAGE analysis

revealed one distinct spot around the marker region at 25-35 kDa. The mass spectra of tryptic digest of the spot indicated positive matches to two homogenous proteins (Table

A-IV). The first protein match from the spot, matched peptides

VASPQQAQEVHVAVR, mass 1617.86 (540.61, 3+), IIYGGSVNGGNSAELAK, mass

1648.84 (825.33, 2+), TRIIYGGSVNGGNSAELAK, mass 1905.99 (636.61, 3+) to TIM

Triosephosphate Isomerase Arabidopsis, a 33 kDa protein (Fig. A-16). MS/MS

fragmentation of VASPQQAQEVHVAVR yielded twenty-five fragment ions of the

*++ 0++ ++ forty-five most intense peaks matching the expected masses b2, b3, b6 , b6 , b7, b8 ,

++ *++ ++ ++ ++ *++ 0++ *++ 0++ ++ b12 , y2, y3, y3 , y4, y5, y5 , y6, y7, y7 , y8, y10 , y10 , y10 , y11 , y11 , y12 ,

++ *++ y13 , y13 . MS/MS fragmentation of IIYGGSVNGGNSAELAK yielded twenty

fragment ions of the fifty-six most intense peaks matching the expected masses b6, b7,

0 * 0 0 ++ * * * b7 , b8, b14, b15, b15 , b15 , b16, b16 , y3, y8 , y9, y10, y10 , y11, y12, y13, y13 , y14, y14 ,

++ *++ 0++ ++ y15, y15 , y15 , y15 , y16 . MS/MS fragmentation of TRIIYGGSVNGGNSAELAK

yielded eight fragment ions of the thirty-four most intense peaks matching the expected

++ * ++ ++ *++ 0++ masses y6, y6 , y6 , y7 , y10, y12 , y14 , y14 . The second protein match from the

spot, matched peptides VASPQQAQEVHVAVR, mass 1616.86 (540.61, 3+),

VAYALSQGLKVIACVGETLEQR, mass 2347.26 (783.64, 3+), to Triosephosphate

Isomerase Lactuca sativa, a 21kDa protein (Fig. A-17). MS/MS fragmentation of

VASPQQAQEVHVAVR yielded thirty-four fragment ions of the fifty-three most

* *++ 0++ ++ ++ intense peaks matching the expected masses b2, b3, b5, b6 , b6 , b6 , b7, b8 , b12 ,

++ * *++ ++ ++ ++ ++ *++ 0++ ++ b13 ,y2, y2 , y3, y3 , y4, y5, y5 , y6, y7, y7 , y8, y8 , y10 , y10 , y10 , y11 ,

*++ 0++ ++ *++ 0++ ++ *++ ++ y11 , y11 , y12 , y12 , y12 , y13 , y13 , y14 . MS/MS fragmentation of

VAYALSQGLKVIACVGETLEQR yielded six fragment ions of the eleven most intense

++ ++ ++ ++ ++ peaks matching the expected masses b6 , b10 , b14 , y2, y17 , y19 .

Soil based Sundance leaf samples 1-D PAGE analysis revealed one distinct spot

around the marker region at 25-35 kDa in the control sample (no EDTA) and one spot in

the EDTA exposed samples. The mass spectra of tryptic digest of the spot indicated

positive matches to two distinct proteins (Table A-V). The peptides observed in the

control spot, SSQSVKSK, mass 721.36 (362.24, 2+), GGSTGYDNAVALPAGGR, mass

1561.75 (781.96, 2+) matched to Oxygen-evolving enhancer protein 1, chloroplast

precursor (OEE1) Arabidopsis, a 35 kDa protein (Fig. A-18). MS/MS fragmentation of

SSQSVKSK yielded eleven fragment ions of the nineteen most intense peaks matching

0 ++ *++ 0++ ++ * ++ 0 ++ the expected masses b3, b3 , b4 , b4 , b4 , b6, b6 , y1 , y2 , y3 , y6 . MS/MS fragmentation of GGSTGYDNAVALPAGGR yielded sixteen fragment ions of the thirty-

*++ 0 0++ * four most intense peaks matching the expected masses b7, b11 , b11 , b11 , b12, b12 ,

0 ++ b12 , b13, b14, b14 , y5, y6, y7, y8, y9, y12. The peptide observed in the EDTA exposed samples matched YGGMVMoIWDR, mass 1095.52 Da (548.75, 2+) to Chitinase H. annuus a 32 kDa protein (Fig. A-19). MS/MS fragmentation yielded twelve and thirteen fragment ions respectively of the sixteen most intense peaks matching the expected masses of b2, b3, b4, b5, b6, b7, y1, y3, y4, y5, y6, y7, y8.

1.3.2. Expression of proteins in Teddy Bear

1.3.2.1. Expression of proteins in response to arsenic and other metals in Teddy Bear

Soil–based Teddy Bear leaf samples 1-D PAGE analysis revealed one distinct spot around the marker region at 25-35 kDa. The mass spectra of the tryptic digest of the spots indicated a positive match to one protein (Table A-VI). The peptides in the spot,

GVMTK mass 534.28 (268.03, 2+), VPADVK mass 627.36 (314.20, 2+) matched to

HSP70 related protein H. annuus, a 28 kDa protein (Fig. A-20). MS/MS fragmentation of

GVMTK yielded two fragment ions of the two most intense peaks matching the expected

++ ++ masses y2 , y4 . MS/MS fragmentation of VPADVK yielded two fragment ions of the

eight most intense peaks matching the expected masses b2, b3.

1.3.2.2. Expression of proteins in response to chromium only in Teddy Bear

Soil–based Teddy Bear leaf samples 1-D PAGE analysis revealed two distinct spots,

one around the marker region at 40 kDa and the other above 200 kDa. The mass spectra

of the tryptic digest of the spots indicated positive matches to one distinct protein in each

spot (Table A-VII). The peptide in the first spot, GVTTIIGGGDSVAAVEK, mass

721.36 (362.24, 2+) matched to cytosolic phosphoglycerate kinase H. annuus, a 42 kDa protein (Fig. A-21). MS/MS fragmentation yielded eight fragment ions of the eleven most

++ intense peaks matching the expected masses b13, y5, y8, y9, y10, y11, y12, y15 . The

peptides in the second spot located past the final marker in the 200+ kDa range,

NILVIGPVPGQK 1233.74 Da (617.93, 2+), QVVDIIPPGPELLVSEGESIK 2218.21 Da

(740.44, 3+) matched to cytochrome f H. annuus a 32 kDa protein (Fig. A-22). MS/MS

fragmentation of NILVIGPVPGQK yielded fourteen fragment ions of the sixteen most

* * ++ intense peaks matching the expected masses b3, b4, b5, b6 , b8, b8 , b11, y4, y6, y7, y7 ,

y8, y9, y10. MS/MS fragmentation yielded nineteen fragment ions of the twenty-nine

* * ++ ++ most intense peaks matching the expected masses b5 , b6, b6 , b12, b14, b15 , b17 ,

*++ 0++ *++ ++ ++ *++ b20 , b20 , y3, y4, y5, y6, y6 , y15 , y16 , y19 .

1.3.2.3. Expression of proteins in response to lead and other metals in Teddy Bear

Hydroponic Teddy Bear leaf samples 1-D PAGE analysis revealed one distinct spot

around the marker region at 40 kDa. The mass spectra of the tryptic digest of the spot

indicated positive matches to two distinct proteins (Table A-VIII). The first set of peptides, VAILGAAGGIGQPLSLLMK mass 18085.06 (905.08, 2+), SSIEKGIKFASQ mass 1293.69 (646.91, 2+), matched to malate dehydrogenase precursor Medicago sativa, a 36 kDa protein (Fig. A-23). MS/MS fragmentation of VAILGAAGGIGQPLSLLMK yielded twenty-one fragment ions of the twenty-six most intense peaks matching the

++ ++ *++ 0++ * expected masses b4, b8, b10, b12, b12 , b15 , b17, b18 , b18 , y2, y3, y4, y7, y8, y8 ,

++ y9, y11, y11 , y12, y13, y15. MS/MS fragmentation of SSIEKGIKFASQ yielded five

0 fragment ions of the ten most intense peaks matching the expected masses b4 , b5, b6,

0 0++ b6 , b8 . The second set of peptides, EPYIGR mass 733.38 (245.02, 3+),

LYSIASSALGDFGDSK mass 1629.79 (815.85, 2+) matched to chloroplast ferredoxin-

NADP+ oxidoreductase precursor Capsicum annuum, a 40 kDa protein (Fig. A-24).

MS/MS fragmentation of EPYIGR yielded two fragment ions of the six most intense peaks matching the expected masses b2, b3. MS/MS fragmentation of

LYSIASSALGDFGDSK yielded twenty-five fragment ions of the thirty-seven most

++ 0 0 0 0 0++ intense peaks matching the expected masses b5 , b5 , b8 , b9 ,b10 , b11 , b12, b13, b14,

* 0 ++ *++ 0++ y2, y3, y5, y5 , y5 , y6, y7, y8, y9, y10, y10 , y11, y12, y14, y14 , y14 .

Hydroponic Teddy Bear stem samples 1-D PAGE analysis revealed one distinct spot below the marker region at 40 kDa. The mass spectra of the tryptic digest of the spot indicated positive matches to two distinct proteins (Table A-IX). The first peptide,

ILIIGGTGYLGK mass 1203.72 (602.95, 2+), matched to NADPH oxidoreductase putative Arabidopsis (Fig. A-25). MS/MS fragmentation of ILIIGGTGYLGK yielded seventeen fragment ions of the nineteen most intense peaks matching the expected

++ 0 0 *++ 0++ 0 0++ masses of b2, b3, b3 , b9 , b10, b10 , y4, y5, y6, y7, y7 , y7 , y8, y8 , y9, y10, y11 .

The second set of peptides, VAILGAAGGIGQPLSLLMK mass 1808.06 (904.92, 2+),

ALEGADVVIIPAGVPR mass 1575.90 (526.32, 3+), DDLFNINAGIVK mass 1317.69

(659.88, 2+), matched to malate dehydrogenase precursor M. sativa a 36 kDa protein

(Fig. A-26). MS/MS fragmentation of VAILGAAGGIGQPLSLLMK yielded twenty-

four fragment ions of the fifty-eight most intense peaks matching the expected masses b7,

* 0 ++ *++ 0++ * 0 0 ++ b10, b11, b12, b12 , b15 , b16, b16 , b18 , b18 , y5, y7, y8 , y9, y9 , y10, y11, y11 , y12 ,

*++ 0++ *++ 0++ ++ y15 , y15 , y17 , y17 , y18 . MS/MS fragmentation of ALEGADVVIIPAGVPR

yielded fourteen fragment ions of the forty-three most intense peaks matching the

++ 0++ ++ ++ ++ expected masses b3, b7, b8, b9, b9 , b9 , b10, b10 , b15 , y4, y6, y6 , y7, y8. MS/MS

fragmentation of DDLFNINAGIVK yielded twelve fragment ions of the twelve most

++ ++ intense peaks matching the expected masses b4 , b7 , b10, b11, y2, y4, y6, y7, y8, y9, y10,

++ y10 .

Hydroponic Teddy Bear leaf samples soluble fraction 1-D PAGE analysis revealed

two distinct spots, one around the marker region at 25-35 kDa and one around the marker

region at 115-125 kDa. The mass spectra of the tryptic digest of the spots indicated

positive matches to one distinct protein in each spot (Table A1-X). The peptide identified in the first spot, YGGMVMoLWDR, mass 1095.52 Da (548.28, 2+) to

Chitinase H. annuus a 32 kDa protein (Fig. A-27). MS/MS fragmentation yielded thirteen fragment ions of the twenty-four most intense peaks matching the expected masses of b2, b3, b4, b5, b6, b7, b8, y3, y4, y5, y6, y7, y8. The peptide identified in the

second spot, AAALAALTSAFNSSSGR mass 1593.81 (797.92, 2+) to villin 3

Arabidopsis a 106 kDa protein (Fig. A-28). MS/MS fragmentation yielded four fragment

++ ++ ++ ions of the five most intense peaks matching the expected masses of y8 , y10 , y15 ,

++ y16 .

Hydroponic Teddy Bear leaf samples transmembrane protein fraction 1-D PAGE

analysis revealed one distinct spot around the marker region at 35 kDa. The mass

spectra of tryptic digest of the spot indicated a positive match to one protein (Table A-

XI). The peptide identified, VINTWQDIINR, mass 1313.71 (657.85, 2+), matched to photosystem II protein D1 H. annuus, a 38 kDa protein (Fig. A-29). MS/MS fragmentation yielded twenty fragment ions of the forty-two most intense peaks matching

* * 0 * * ++ *++ the expected masses b5, b7, b7 , b8 , b8 , b9, b9 , y4, y5, y5 , y6, y7, y8, y9, y9 , y9 ,

0 0++, *++ 0++ y9 , y9 , y10 , y10 .

1.3.2.4. Expression of proteins in response to lead and other metals 2D Gel Separation in

Teddy Bear

Hydroponic Teddy Bear leaf samples 2-D PAGE analysis revealed three distinct spots,

two around the marker region at 25-35 kDa, 4 – 5 pH, and one around the marker region

at 45 kDa, 5-7 pH. The mass spectra of the tryptic digest of the spots indicated positive

matches to one protein in the first spot, two distinct proteins in the second spot and one

protein type in the third spot (Table A-XII). The peptides identified in the first spot,

RPHAVLINSISSSR, mass 1535.85 (769.78, 2+), NFRLKDLPRLIK, mass 1511.93

(757.49, 3+), NPNDLTIRFNTEVADKCLR, mass 2218.12. (740.27, 3+) matched to

Cytokinin-O-glucosyltransferase M. trunculata, a 31 kDa protein (Fig. A-30). MS/MS

fragmentation of RPHAVLINSISSSR yielded seven fragment ions of the seventeen most

++ ++ *++ 0++ intense peaks matching the expected masses b9 , b12 , y3, y6, y6 , y6 , y7. MS/MS

fragmentation of NFRLKDLPRLIK yielded six fragment ions of the ten most intense

* ++ peaks matching the expected masses y4, y5 , y6, y7, y7 , y10. MS/MS fragmentation of

NPNDLTIRFNTEVADKCLR yielded seven fragment ions of the seven most intense

* ++ ++ *++ 0++ ++ ++ peaks matching the expected masses b3 , b10 , b17 , y5 , y5 , y8 , y15 . The first

set of peptides identified in the second spot, KHHLFG, mass 737.40 (737.59, 1+),

AQKPVHDASKR, mass 1235.67 (619.31, 2+) matched to ABA/WDS stress induced

protein O. sativa, a 24.5 kDa protein (Fig. A-31). MS/MS fragmentation of KHHLFG

yielded three fragment ions of the thirteen most intense peaks matching the expected

masses b3, b4, b5. MS/MS fragmentation of AQKPVHDASKR yielded four fragment

++ ++ ++ ions of the nineteen most intense peaks matching the expected masses y5 , y6 , y8 ,

++ y10 . The second set of peptides identified in the second spot, GRGLLVGMK, mass

929.55 (466.22, 2+), GEKTPWMHEYR, mass 1531.72 (766.64, 2+) matched to NAC

domain protein O. sativa, a 34 kDa protein (Fig. A-32). MS/MS fragmentation of

GRGLLVGMK yielded two fragment ions of the three most intense peaks matching the

expected masses b2, b6. MS/MS fragmentation of GEKTPWMHEYR yielded four

++ fragment ions of the nineteen most intense peaks matching the expected masses y5 ,

++ ++ ++ y6 , y8 , y10 . The peptides in the third spot matched 1-aminocyclopropane-1- carboxylic acid oxidase from two different species, DFQDAIK, 835.41 mass (179.08,

3+), VYGPIK, mass 675.40 (226.11, 3+), GLGGTSALLDFRV, mass 1107.59 (639.30,

2+), matched to Arabidopsis (Fig. A-33) and AIANFMSKAR, mass 1276.70 (370.23,

3+), matched to Rosa hybrid cultivar (Fig. A-34). MS/MS fragmentation of DFQAIK

yielded three fragment ions of the three most intense peaks matching the expected masses

b2, b3, y3. MS/MS fragmentation of VYGPIK yielded five fragment ions of the twelve

++ ++ * ++ ++ most intense peaks matching the expected masses b3 , b4 , y2 , y3 , y4 . MS/MS

fragmentation of GLGGTSALLDFRV yielded eleven fragment ions of the twenty-nine

0 0 0++ ++ ++ ++ most intense peaks matching the expected masses b5 , b7 , b12 , y3 , y4 , y5, y6

++ *++ ++ ++ y8 , y8 , y10 , y12 . MS/MS fragmentation of AIANFMSKAR yielded three

++ ++ fragment ions of the four most intense peaks matching the expected masses b3 , b5 ,

++ b6 .

Hydroponic Teddy Bear leaf samples 2-D PAGE analysis revealed four distinct spots,

two around the marker region at 25-35 kDa, 4 – 5 pH, one around the marker region at

80-90 kDa, 5-7 pH and the last around the marker region at 25-35 kDa , 8-10 pH (Fig. 1-

4). The mass spectra of the tryptic digest of the spots indicated positive matches to two

distinct proteins in three spots, and one distinct protein in the fourth spot (Table 1-II).

The first peptide identified in the first spot, YGGMVMoLWDR, mass 1095.52 Da

Figure 1-4. 2-D Gel Separation Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb

(in submission and under review: Atkinson, I., Hasintha, C., vanKeulen, H., Cutright, T., Wei, R. Proteomic analysis of soluble proteins induced by toxic metals in Helianthus annuus. (Journal of Proteomics))

Table 1-II. 2-D Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb Marker Region Theoretical Peptide kDa Mass kDa pH Accession no. sp Protein Score (Coverage) Peptides

25-35 32 4-5 gi|126723930 H. annuus Chitinase 62 1(2%) YGGVMIWDR jacalin - like protein 25-35 32 4-5 gi|21633213 H. vulgare LEM2 24 1 (3%) SDVTLKALGVYVK Solanum Putative thaumatin- 25-35 23 4-5 gi|53830843 tuberosum like protein 28 1(4%) TQGGCNNPCTVYK hevein-like E. antimicrobial 25-35 35 4-5 gi|30844168 europaeus peptide 19 1 (2%) SRNPQVEDR

TTPSYVAFTDTER, NQVAMNPQNTVFDAK, Heat Shock 70 kDa SQVHEVVLVGGSTR, 80-90 70 5-7 gi|18663 Glycine max protein 41 4 (9%) FELTGIPPAPR TTPSYVAFTDTER, IINEPTAAAIAYGLDK, HSP 70B; ATP FEELNMDLFR, 80-90 71 5-7 gi|15219109 Arabidopsis binding 41 4 (9%) NTIKDEK

20-35 32 8-10 gi|42573009 Arabidopsis metal ion binding 32 1 (3%) CCKGCQTKAKR

(A)

MEFTHPALLLLLFITVFSFLKPSTAAGIATYWGQQSDDTEGTLEAACATGNYQFV

NIAFLSTFGNNQQPVLNLAAHCDPASTCSRYSSQIKACQAQNVKVFLSIGGQRGS

YSLSSPQDAQQVADFLWNTYLGGQPATRPLGDAVLDGIDFDIEQGTDQFWSDLA

KAPAAYSSQKKVYLSAAPQCPFPSGDVRNQLLPAIREGLFDYVWVQFYNNEQCQ

YGANADALLARWNEWTQVTTNTIFLGLPAAASGAAPSGGYIPPDILTSQILPSIKS

SPKYGGVMLWDRFYDKQSGYSDAIKGSIN

(B)

Figure 1-5. Tandem mass spectra of polypeptide (A) from Chitinase (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

(548.75, 2+) to Chitinase H. annuus a 32 kDa protein (Fig. 1-5). MS/MS fragmentation

yielded eleven fragment ions of the sixteen most intense peaks matching the expected

masses of b2, b3, b6, b7, y1, y3, y4, y5, y6, y7, y8. The second peptide identified in the first spot, SDVTLKALGVYVK, mass 1391.16 Da (696.59, 2+) matched to jacalin-like protein LEM2 H. vulgare, a 32 kDa protein (Fig. A-35). MS/MS fragmentation yielded

0 ten fragment ions of the twenty-six most intense peaks matching the expected masses b3 ,

++ ++ ++ ++ *++ ++ ++ y4, y4 , y6, y6 , y8 , y9 , y10 , y11 , y12 . The first peptide identified in the second

spot, TQGGCNNPCTVYK, mass 1499.01 Da (750.54, 2+) matched to thaumatin-like

protein S. tuberosum, a 25 kDa protein (Fig. 1-4). MS/MS fragmentation yielded eight

fragment ions of the twenty-two most intense peaks matching the expected masses b5,

* ** ** 0 *++ 0++ b5 , b7, b7 , b8 , y10 , y11 , y11 . The second peptide in the second spot,

SRNPQVEDR, 1099.38 Da (550.70, 2+) matched to hevein-like antimicrobial peptide,

E. europaeus, a 32 kDa protein (Fig. A-36). MS/MS fragmentation yielded three

fragment ions of the seven most intense peaks matching the expected masses y3, y4, y6.

The first set of peptides in the third spot, TTPSYVAFTDTER, 1486.44 Da (744.23, 2+),

NQVAMNPQNTVFDAK, 1676.68 Da (839.35, 2+), IINEPTAAAIAYGLDK, 1658.56

Da (830.29, 2+), FEELNMDLFR, 1332.34 Da (667.18, 2+), SQVHEVVLVGGSTR,

1465.76 Da (733.89, 2+), FELTGIPPAPR, 1196.48 Da (599.25, 2+), NTIKDEK, 848.14

Da (425.08, 2+) matched to heat shock protein 70, Arabidopsis, a 70 kDa protein (Fig. A-

37). MS/MS fragmentation of TTPSYVAFTDTER yielded eighteen fragment ions of the

* 0 * thirty-three most intense peaks matching the expected masses b4, b9, y3 , y3 , y4, y5, y5 ,

0 ++ ++ *++ 0++ y6, y7, y7 , y8, y9, y9 , y10, y11, y11 , y11 , y11 . MS/MS fragmentation of

NQVAMNPQNTVFDAK

(A)

MHFLKFFPLFVFLYFGQYYLYVTHAATFDITNRCTYPVWAAASPGGGRRLDSGQ

TWNLNVNPGTIQARIWGRTNCNFDGSGRGKCETGDCNGLLECQGYGSPPNTLAE

FALNQPNNLDFVDISLVDGFNIPMEFSPINGGCRNLLCNAPINDQCPNELRTPGG

CNNPCTVFKTNEFCCTNGPGSCGPTDLSRFFKQRCPDAYSYPQDDPTSLFTCPAG

TNYKVVFCP

(B)

Figure 1-6. Tandem mass spectra of polypeptide (A) from thaumatin-like protein (S. tuberosum) as indicated (bold) in the primary amino acid sequence (B).

yielded twenty fragment ions of the thirty-seven most intense peaks matching the

* * ++ * *++ 0 expected masses b4, b5 , b6, b11, b13, y4, y6 , y7, y8, y9, y9 , y10, y11, y11 , y11 , y11 ,

0++ *++ y11 , y12, y12 , y13. MS/MS fragmentation of IINEPTAAAIAYGLDK yielded eleven

fragment ions of the twenty-six most intense peaks matching the expected masses b3,

++ ++ *++ 0++ *++ 0++ ++ *++ *++ b5 , b8, b9 , b11 , b11 , b14 , b14 , b15 , b15 , y11 . MS/MS fragmentation of FEELNMDLFR yielded four fragment ions of the eleven most intense peaks matching

0 the expected masses b2, b5, b5 , b8. MS/MS fragmentation of SQVHEVVLVGGSTR

yielded nine fragment ions of the thirty-five most intense peaks matching the expected

++ * ++ masses b8 , y5, y5 , y6, y7, y8, y10, y11, b13 . MS/MS fragmentation of FELTGIPPAPR

yielded ten fragment ions of the thirty most intense peaks matching the expected masses

0 0++ b2, b4, b6, b6 , b9 , b10, y5, y7, y8, y9. MS/MS fragmentation of NTIKDEK yielded six

++ ++ fragment ions of the eight most intense peaks matching the expected masses b4 , b5 ,

++ *++ 0++ ++ y2 , y4 , y4 , y5 . MS/MS fragmentation of FELTGIPPAPR yielded ten fragment

0 0++ ions of the thirty most intense peaks matching the expected masses b2, b4, b6, b6 , b9 , b10, y5, y7, y8, y9. The second set of peptides in the third spot matched to RuBisCo, a

protein whose strong band is evident in every gel, and seen as a co-contributing protein in

many analyses. The peptide identified in the fourth spot, CCKGCQTKAKR, 1223.28 Da

(612.65, 2+), matched to a metal ion binding protein, Arabidopsis, a 30 kDa protein (Fig.

A-38). MS/MS fragmentation yielded four fragments of the eight most intense peaks

matching the expected masses b3, b4, b5, b7.

1.4. Discussion

An abundance of defense and stress induced proteins have been identified within the

samples analyzed. Protein families have been highlighted, and in many cases these

proteins have a primary role associated with their function, and a seeming secondary or

surveillance role involving stress tolerance. In our study we have seen proteins

associated with biotic stress including mechanical wounding, bacterial, viral and fungal

infection; and also with abiotic stress, such as drought, light, ABA, temperature, osmotic

pressure and toxic chemicals. These defense/stress response proteins are responsible for

maintaining proper cell function and homeostasis, implying the need for roles in

maintaining cell structure and integrity, resource allocation and usage, material transport

and storage, and repair mechanisms. To see such a wide array of stress response proteins

exhibited in response to metal stress implies a more universal set of stress related

functions of the proteins expressed, as opposed to many more specific responses

observed from some proteins to individual stresses.

Chitinase, identified in samples from both Sundance and Teddy Bear cultivars, in roots and leaf tissues, exposed to both As and Pb in conjunction with Cd, Cr and Ni, enzymatically catalyzes the degradation of chitin, a linear ß1-4-linked polymer of N- acetyl-D-glucoseamine (GlcNAc). Chitinases are expressed by stress such as fungal and bacterial infection, and are also documented to be expressed under other stress conditions such as heavy metal exposure. Chitinases were positively determined to be up-regulated

on the genetic level by As exposure using RT-PCR expression (Fig 1-7B, lane 6 & 7) and

western blot analysis in H. annuus (Fig 1-8, Lane 3,4 & 8) [11]. This study described the

isolation and determination of chitinase in plants exposed to As alone and in combination

Figure 1-7. Semi-quantitative RT-PCR of total RNA samples showing an upregulation of Chitinase in samples containing As [11].

Figure 1-8. Western Blot analysis of Sundance and Teddy Bear samples showing an up-regulation of Chitinase in samples exposed to As [11].

with Cd, Cr, and Ni. In addition chitinase has now also observed to be up-regulated in

the presence of Pb alone and in combination with Cd, Cr, and Ni.

Oxygen-evolving enhancer protein 1 (OEE1) (match to Arabidopsis), thylakoid membrane protein, was identified from Sundance leaves, upon exposure to Pb, Cd, Cr,

and Ni. No OEE proteins has been described in the NCBI database for any species of

Helianthus. This protein shares similarity with the proteins attributed to stabilize the

manganese cluster which is the primary site of water splitting in the choloroplast, and is a

subunit of the photosystem II complex. This protein has been documented to be

increased under salt stress conditions in Bruguiera gymnorrhiza [19] to compensate for the depression of photosynthesis. This protein has also been found to exhibit thioredoxin activity in green algae [20], a possible mechanism to deal with environmental stresses on the photosystem II complex. The OEE1 protein has been attributed with regulating light induced oxidative damage to the photosystem II complex and maintaining the efficient turnover of the damaged subunit [21]. OEE2, an oxygen evolving complex protein, has also been documented to have increased expression in Vigna unguiculata under Mn stress

[20].

An amino terminal coiled-coil, nucleotide-binding site containing, leucine-rich repeat resistance gene analog (CC-NBS-LRR RGA) protein was identified from Sundance roots exposed to As, Cd, Cr and Ni. These proteins are typically expressed in plants as a triggered defense response to a broad range of disease conditions. In addition to pathogen related expression a CC-NBS-LRR protein has been documented to be upregulated under water stress, to confer significant drought tolerance in Arabidopsis

[22].

Lectin (H. tuberosus) was identified from Sundance roots exposed to As, Cd, Cr, Ni.

No lectins exist in the database for H. annuus. Lectins are a group of proteins classified

by their recognition and binding specificity to carbohydrate ligands that are widely

distributed in higher plants and are believed to play a role in plant defense [23, 24, 25].

The family of jacalin-related plant lectins is mannose binding, specific for Man-α-1-

3Man and Man-α-1-2Man, which are abundant in the glycoconjugates exposed on the

surface of viruses, bacteria, and fungi and on the epithelial cells along the gastrointestinal

tract of lower animals [26]. This specificity may allow these lectins to contribute to plant

stress and defense response to pathogens. The jacalin-related lectins are composed of 1-6

copies of the jacalin-like lectin domain, a small 15 kDa domain that can explain why the

protein was identified from a band in the 60 kDa range. Lectin is also observed to be upregulated in the presence of Pb in combination with Cd, Cr, and Ni.

Actin was identified in Sundance leaves exposed to As alone, not in combination with any other metals. Actin is a highly conserved eukaryotic protein and one of the most abundant proteins in a eukaryotic cell. Actin is associated with cell structure and various cell maintenance functions thru interaction with a large number of Actin-binding proteins. Actin has been reported as up-regulated in Cannabis sativa under copper stress

[16]

Glyceraldehyde 3-phosphate dehydrogenase B subunit (GAPDH) (Arabidopsis) was

identified in Sundance leaves exposed to As alone, not in combination with any other

metals. The peptides observed and the protein identified share homology with H. annuus

GAPDH. GAPDH catalyzes the sixth step of glycolysis, playing a primary role in plant

metabolism. This protein has also been identified as an anti-stress protein due to its interaction with cytosolic thioredoxins [27].

Glycolate oxidase (GOX) (Arabidopsis) was identified in Sundance leaves exposed to

As alone, not in combination with any other metals. There are no glycolate oxidase proteins in the database for Helianthus. GOX is a key photorespiratory enzyme, carrying out the oxidation of glycolate to glyoxylate. Its activity was documented to increase due to osmotic stress [28], salt stress [29] and drought stress [30].

Photosystem II protein D1 was identified in Sundance and Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. Photosystem II is the site of water oxidation in photosynthesis and is particularly susceptible to damage by light stress [21]. This proteins upregulation is observed in response to stress damage do to turnover and is closely associated with

OEE1. [31]

Triosephosphate isomerase (TIM, Arabidopsis) was identified in Sundance and Teddy

Bear leaves exposed to Cd, Cr, Ni, and Pb. TIM reversible interconverts dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate in glycolysis and gluconeogensis [1]. This is a general housekeeping enzyme found in all cells; no association of this enzyme with metal stress has been previously reported.

Hevein-like antimicrobial protein (Euonymus europaeus) was identified in Sundance and Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. There are no hevein-like proteins in the database for Helianthus. Hevein-like proteins are small proteins with chitin binding activity associated with the lectin super family. These proteins have antifungal activity, even against non-chitinous fungus, and are expressed in response to microbial

infection in plants [32, 33]. This activity suggests additional pathogen resistance

properties associated with the protein in addition to its known chitin-binding function.

ATP synthase CF1 alpha subunit was identified in Sundance leaves exposed to Cd, Cr,

Ni, and Pb. ATP synthase hydrolyzes ATP within the chloroplast and is driven by the

electron-transport process. ATP synthases are up-regulated under osmotic and stress conditions [23,34]. ATP synthase activity is reduced under Fe2+ and oxidative stress

conditions due to oxidative modification of amino acid residues required for proper

function [35]

Drought-induced protein SDi-6 (designated sdi for sunflower drought induced) was

identified in Sundance leaves exposed to Cd, Cr, Ni, and Pb. SDi-6 is a water stress induced protein whose presence is associated with an increase in plant tolerance to drought conditions [36]. SDi-6 has no matching plant family domain in the NCBI database although drought induced proteins are also associated with ABA stress as abscisic acid levels rise in low water conditions [36].

Glucosyltransferase-like protein (A. thaliana) was identified in Sundance leaves exposed to Cd, Cr, Ni, and Pb. There are no protein matches or homologies to glucosyltransferase proteins in the database for Helianthus. This metabolic protein type

is documented to be upregulated in response to ABA, drought, salt and cold stress [37].

Quinone oxido-reductase-like was identified in Sundance stems exposed to Cd, Cr, Ni,

and Pb. Quinone oxido-reductase (QOR) is regulated by the aryl hydrogen receptor

complex as a detoxification mechanism for many toxic hydrocarbon compounds. QOR

has been documented to be up regulated in response to Ar, Cd, and Cr exposure in mouse

hepatoma Hepa 1c1c7 cells studied [38].

Phenylcoumaran benzylic ether reductase (PCBER) homolog TH3 (T. heterophylla) was identified in Sundance stems exposed to Cd, Cr, Ni, and Pb. There are no homologous proteins in the Helianthus database. PCBER is part of a class of related enzymes involved in the production of plant defense compounds in response to fungal stress. These related enzymes and their products are found across the entire plant kingdom and suggest the possibility of an underlying universal defense response [39].

Isoflavon reductase-homologue (IFR) (N. tabacum) was identified in Teddy Bear stems and leaves exposed to Cd, Cr, Ni, and Pb. There are no homologous proteins in the database for Helianthus. Phylogenetically related to phenylcoumaran benzylic ether reductase. IFR also plays a role in plant defense to fungal infection thru the production of antimicrobial phytoalexins [39].

Malate dehydrogenase (MDH) (M. sativa) was identified in Sundance and Teddy Bear stems and leaves exposed to Cd, Cr, Ni, and Pb. There are no MDH proteins in the database for Helianthus. MDH has an antioxidant role in cell defense through its conversion of malate to oxaloacetate, an ROS scavenging specie [40]. MDH has also been identified as an anti-stress protein due to its interaction with cytosolic thioredoxins

[27].

Chloroplastic drought-induced stress protein (CDSP-34) was expressed in Teddy

Bear leaves exposed to Cd, Cr, Ni, and Pb. CDSP-34 is a member of the plastid associated protein (PAP) fibrillin family. There exists no match in the NCBI protein database for this protein or family (PAP fibrillin super family) to any Helianthus species.

CDSP-34 is documented to be expressed in response to many stresses; drought, high light exposure, oxidative, bacterial infection and mechanical wounding [41-44].

Heat-shock protein 70 (HSP-70, Arabidopsis) was expressed in Teddy Bear leaves exposed to As, Cd, Cr, and Ni and Pb, Cd, Cr, and Ni. HSP’s are responsible for protein folding, assembly, translocation and degradation in many normal cellular processes.

HSP’s stabilize proteins and membranes, and can assist in protein refolding under stress conditions [45]. Hsp70 protein acts as a molecular chaperone binding to hydrophobic residues exposed by stress conditions, preventing proteins from agglomerating and helping to refold these denatured proteins. Hsp70 also assists in protein transport across membranes by its hydrophobic interaction, assisting to unfold, stabilize and refold proteins during transport. [45,46]. Members of the HSP 70 family are upregulated as a result of thermal and oxidative stress including heavy metal exposure [47]. HSP-70 was also identified as an anti-stress protein due to its interaction with cytosolic thioredoxins

[27].

Thaumatin-like protein, a class 5 pathogensis-related proteins, were expressed in

Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. Thaumatins are expressed in response to fungal and viral stresses as well as abiotic stress factors such as increased salinity and drought [48- 50]. Many thaumatin-like proteins also bind to ß1,3-glucans and also display endo- ß 1,3-glucanase activity, as associated with antimicrobial resistance [51]. A

Thaumatin-like protein has been positively documented to be up-regulated in response to

Pb exposure using RT-PCR expression in H. annuus (performed by Dr vanKeulen, results not shown).

Chloroplast ferredoxin-NADP+ oxidoreductase precursor (FNR) (C. annuum) was expressed in Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. There are no homologous

proteins in the database for Helianthus. FNR plays an antioxidant stress response role in

cellular defense against oxidative damage [52, 53].

Villin 3 (Arabidopsis) was expressed in Teddy Bear leaves exposed to Cd, Cr, Ni, and

Pb. There are no homologous proteins in the database for Helianthus. Villin is an actin

binding, bundling, severing, nucleating and capping protein whose function is regulated

by calcium binding and is expressed ubiquitously in Arabidopsis [54].

Cytokinin-O-glucosyltransferase (M. trunculata) was expressed in Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. There are no homologous proteins in the database for

Helianthus. Cytokinin-O-glucosyltransferases glycosylate cytokinins, plant hormones essential for growth and development and that are also documented to provide protection

against degradative enzymes [55]. Cytokinin-O-glucosyltransferase has also been documented to be upregulated in leaves of plants under drought stress [56].

Abscisic acid, water deficit (ABA/WDS) stress induced (Orytza sativa) was expressed

in Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. No homologous proteins exist in the

database for Helianthus. This is a family of proteins is induced by abscisic acid stress

and drought/water deficit stress (WDS) and also has a noted upregulation under ripening

[57]. The proteins role and function is not well understood.

NAC Domain protein (H. annuus) was expressed in Teddy Bear leaves exposed to Cd,

Cr, Ni, and Pb. NAC Domain proteins are a family of transcription factors that have been

observed to be upregulated in response to biotic and abiotic stressors, specifically cold,

salt, water and wounding [58] as well as pathogen recognition and defense [59].

1-aminocyclopropane-1-carboxylate oxidase (ACO) (Arabidopsis) was expressed in

Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. ACO converts 1-aminocyclopropane-

1-carboxylate to ethylene, a hormone in plants that plays a role in development, and

response to stress and pathogen defense [60]. The ethylene plant stress-response pathway

is induced by mechanical wounding, chemical, metal, drought, temperature stresses [61].

Jacalin - like protein LEM2 (H. vulgare) was expressed in Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. No homologous protein exists in the database for Helianthus, although this protein does share some family homology to the lectin identified in

Sundance leaves exposed to Pb, Cd, Cr, Ni. Jacalin-like proteins are a class of lectins that bind carbohydrates and are expressed in response to abiotic and biotic stresses [62,

63].

Heavy-metal-associated domain-containing protein (HMA) (Arabidopsis thaliana) was

expressed in Teddy Bear leaves exposed to Cd, Cr, Ni, and Pb. No homologous protein

exists in the database for Helianthus. HMA domain containing proteins are known as

transport and detoxification proteins. HMA proteins in addition to metal stress have been

shown to be upregulated under drought, light, temperature and ABA stressors [64].

Cytosolic phosphoglycerate kinase (CytPGK) (H. annuus) was identified in Teddy

Bear leaves exposed to Cr. CytPGK is a glycolysis enzyme that transfers a phosphate

group from 1,3-bisphosphoglycerate to ADP, forming ATP. CytPGK has been shown to

be upregulated under cold stress conditions [65].

Cytochrome f (H. annuus) was identified in Teddy Bear leaves exposed to Cr.

Cytochrome b6f is the electron/proton transfer protein complex of oxygenic

photosynthesis that functions between photosystems II and I. Cytochrome f, the largest

subunit of the b6f complex, consists of an N-terminal 251-residue redox-active domain in

the lumen-side aqueous phase, and a 35-residue C-terminal transmembrane anchor in the

photosynthetic membrane of Chlamydomonas reinhardtii [66]. There are no citable articles implying cytochrome f upregulation due to stress.

1.5. Conclusions

The expression of many proteins are regulated by biotic and abiotic stresses,

suggesting the occurrence of complex mechanisms that control gene expression in

response to environmental stresses. Similarly, evidence exists for crossed response by

different stimuli [69]. It is well established that many environmental stimuli such as

salinity, drought, heat or cold, elicit common stress responses in up-regulated proteins

and that these proteins share a general role of protecting the plant from damage and

repairing damage already incurred [70]. The proteins observed in H. annuus as a result

of heavy metal exposure follow this response, as stress related proteins involved in

defense and repair mechanisms, although not individually specific to heavy metal

binding. This shared functionality of proteins under various stress condition is favored

by evolution theory, which encourages the re-use and adaptation of existing proteins for

multiple functions.

Proteins identified in the analysis encompass a wide role, including those classified

with metabolic function, photosynthesis, general cellular maintenance, antimicrobial

response, drought and water stress response, general stress and antioxidant roles. These potentially expressed proteins may play a role in rendering plants tolerant of these toxic metals, thus enabling the plants to hyperaccumulate them. Understanding how these proteins function in response to metal stress and determining how their roles confer stress tolerance will allow a greater application and effective implementation of phytoremediation.

The LC-MS identification of potentially expressed proteins still requires a need to set the bar for positive identification of a proteins gene upregulation. Coupling a matched peptide from the MASCOT database to a gene sequence for a protein allowed for the design of a primer and the expression of the gene for RT-PCR analysis. Further generation of an anti-serum to the determined protein sequence allowed Western Blot analysis. Using these techniques a positive upregulation of Chitinase in root and leaf tissues exposed to As and Pb alone and in combination with Cd, Cr, and Ni in H. annuus has been documented. Additionally a Thaumatin like proteins up-regulation in leaf tissues exposed to Pb in combination with Cd, Cr, and Ni in H. annuus has been

documented.

Future work will be directed to addressing several issues this study has brought to

light. First is the doubt of positive identification via LC-MS and bioinformatics alone. A

protein identification based on a single peptide will always leave a question as to the

confidence. Additional sequence coverage, even just a second tryptic peptide match,

greatly increases confidence in the likelihood of correlation to the database resolved

protein. To increase sequence coverage analysis methodology can be refined, shifting to

a nano-flow analysis alone will greatly increases mass spectrometric results. Cross sequence coverage can also be performed using additional digest parameters with alternative enzymes which also allows for a very strong confidence even in the case of very limited sequence coverage. The second problem is the bioinformatics database itself. Protein identifications made are highly determined by the skill of the user in interpreting the results of the mass spectrum analyzed and properly filtering the database results. With the sheer majority of organisms’ protein libraries being far from complete

the likelihood of fit to other species informatics need to be considered. Oftentimes an

understanding of protein function and structure is needed, as some initial search results

may not seem relevant or match the gel data determined (mass and P.I.) and be prematurely removed from possible matches. Finally there is high conservation of many peptide motifs across a wide range of proteins and protein families which can force it to the user’s interpretation to determine a “goodness of fit” for a correct match. Further development of the databases utility and an increase in the repository of protein information will help resolve this issue.

In summary, the versatility of methodology, wide range of application and near limitless combinations of chromatography and mass spectrometry techniques 2D LC-Ion

Trap MS/MS coupled with MASCOT bioinformatics is a powerful tool available to the biochemical researcher. The ability to tackle large mixtures of proteins and molecules, with basic preparation is an amazing feat of modern scientific application. Further “de novo” sequencing to determine identity of proteins unknown to the database can be performed by varying additional digest methods and conditions to increase the total peptide identification, elucidate secondary structure, and improve confidence. Separation gradients and column specifications can be refined with repetitive analysis to tailor fit each samples unique make-up. Additional steps can be incorporated to further identify or isolate particular peptides or proteins; such as the incorporation of affinity columns or size exclusion steps in series with or in place of the CapTrap. Peptide mass mapping with the sensitivity of the ion trap coupled with collision-induced dissociation spectra allows for an incredibly detailed picture of a protein to be created, including identification of posttranslational modifications and single amino-acid variations. Even

whole protein complexes are able to be studied with ESI-ion trap methodology, expanding the technique beyond protein identification to exploring protein function and interaction. In all, this technique stands on the forefront of a rapidly expanding field of

Proteomics wherein it seems the only limitation is the imagination of the BioMolecular

Chemist.

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deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene

under 35S or SAG12 promoters. Plant, Cell and Environment 31:341-353

57. Wang, J.-T., Gould, J.H., Padmanaghan, V., Newton, R.J. (2003) Analysis and

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CHAPTER II

STUDY ON ADDUCTION REACTIONS IN

CHYMOTRYPSIN’S ACTIVE SITE

2.1. Background and Significance

PAHs, composed of hydrogen and carbon, are hydrophobic pollutants made up of two

or more fused benzene rings. In addition, some may contain alkyl and other substituent

groups. They are formed as the byproducts of the pyrolysis and incomplete combustion

of organic matter [1]. In industrialized countries, the major sources of PAHs are

petroleum products such as oil, coal, tar, creosote, and vehicle emissions [2], though they

are also formed as byproducts of energy usage, heating, and cooking [3]. Human exposure is predominantly through breathing, ingestion, dermal contact, and cigarette smoke [4]. Though inherently found in the environment as a consequence of natural phenomena (e.g. volcanic eruptions, forest fires) their presence has greatly increased since the advent of industry, the mainstream use of fossil fuels as power and heat sources, the development of the combustion engine, and the grand-scale deforestation of natural habitats. The thermal stability of PAHs coupled with the wide array of potential sources has lead to the ubiquitous nature of the pollutants’ dispersal; the range of concentration of

BaP throughout the environment is shown in Fig. 2-1.

Figure 2-1. Conc. of Benzo[a]pyrene detected in air, soil, water, and plants and food

[3].

Research has been undertaken to qualify and quantify the carcinogenic/mutagenic nature of PAHs. In an attempt to utilize the protein-PAH adduct as a biomarker, blood proteins from industrial workers as well as urban and rural residents of many industrialized countries have been evaluated, with correlating studies being conducted on control patients exposed to similar contaminant levels [5-14]. Attempts to correlate the level of PAH exposure to cancer rates and tumor growth in lab animals have also been conducted, yielding a positive correlation between PAH exposure and tumor development [15]. Of these studies the focus on protein-PAH adduct formation has primarily been on hemoglobin and albumin; due to the vast amount of literature present, the ease of isolation and their large quantity per gram of blood.

The qualification of PAH toxicity has been attributed to the PAHs’ metabolic cycle in the body. Examination of the biochemical degradation steps of the PAHs’ metabolism in humans have found that CPY1A1 is the principle enzyme involved in their initial monooxygenation [3]. This enzyme is responsible for the PAHs’ “activation” to an electrophilic species capable of reacting with nucleophilic sites on DNA or Protein. The various metabolites of the CPY1A1 reaction with BaP can be seen in Fig. 2-2.

Though research into the effects that the PAH – DNA adduct has on helix structure and its subsequent isolation could undeniably pin point genetic mutation/mistranslation events there are two distinct drawbacks to studying the DNA adduct. Firstly, DNA inherently has a specific repair mechanism not present in proteins that may allow for adduct removal and proper helix restoration. Secondly the vast amount of intron and satellite DNA in exposed cells makes risk assessment of toxins very difficult to quantify.

The seemingly rare possibility of an adduct formation event occurring within an exon as

Figure 2-2. Metabolism of Benzo [a] Pyrene [1].

opposed other “non-transcribed/translated” genetic material makes the immediate mutagenicity of the DNA-PAH adduct difficult to correlate with abnormal cellular function.

The protein adducts that are formed upon contact with the CPY-1A1 altered PAH in the cellular or extra-cellular environment lack a specific repair mechanism and allow for a decisive determination of, if any, altered proteolytic function. These altered proteins can still be involved in biochemical reactions, only now in a non-homologous state with their unaltered forms for the remainder of their “lifetime”. The effects that these alterations may have on the proteins kinetics and structural conformation are the objectives that this research hopes to address.

This study will follow suit with past work done in the preparation and purification of protein-PAH adducts and proposes a next step into understanding protein alterations.

Past research has focused on two separate methods for determining adduct formation.

The initial research focused on determining adduct placement on the primary amino acid sequencing of the protein by lysing the denatured protein and piecing together the observed fragments [16-17]. Further methods have isolated the specific residues involved in adduct formation by hydrazinolysis [18-20]. The end goal of these studies was to quantify the level of adduct formation with the subjects exposure, again attempting to utilize the protein adduct as a “bio-marker”.

To additionally qualify an adducts effect on protein function an analysis of protein kinetics will be performed. For this analysis a good protein model needs to be chosen.

Contributing to this are the findings of prior experimentation into protein-PAH adduct formation that revealed the high affinity of BaP diol epoxides for the imidazole nitrogen

of histidine residues [16–22], and the potential affinity that electrophiles have in nucleophilic substitution reactions within receptor binding sites [23]. This alkylation

affinity of the metabolically activated PAH leads to the choice of chymotrypsin (CHY)

for the study. CHY, a member of the serine peptidase class of enzymes, has a catalytic

core whose functionality is based in part on a centrally located histidine residue Fig 2-3.

The high potential for disruptions of the natural function of these proteins by PAH adduct

formation with the catalytic core’s histidine residue makes this an excellent enzyme for

analysis.

Figure 2-3. Catalytic core of Chymotrypsin, a model Serine Peptidase.

To determine and optimize conditions for CHY-PAH adduct analysis a known CHY- adduct will be assessed, the irreversible inhibition of chymotrypsin by exposure to tosyl phenylalanyl chloromethylketone (TPCK). TPCK irreversibly inhibits CHY by binding to histidine 57 located in the center of CHYs active sites catalytic triad (Fig. 2-4). This

reaction is specifically mediated by CHY’s hydrophobic pocket located adjacent to its

active site. This pocket facilitates the placement and orientation of the aromatic side

groups on TPCK, much as in CHY’s normal enzymatic function. When oriented in the

hydrophobic pocket TPCK’s chlorinated methyl group reacts with the active site histidine. The pocket’s hydrophobic character and facilitation of substrate orientation for nucleophilic substitution reaction is potentially a viable course of reaction after exposure of the serine proteases to PAH metabolites.

Figure 2-4. TPCK reaction/inhibition of CHY.

This hydrophobic pocket on CHY has also been studied using a theoretical software program for “goodness of fit” for a large group of hydrophobic compounds as reversible inhibitors [28]. This work is of interest in applying the effects of PAH and PAH metabolites on CHY kinetics in a real time experiment. Preliminary trials of CHY +

PAH solutions had some interesting effects on absorbance data, particularly the 2 and 3 ring species, and their reduction of CHY Kinetics.

Additional theoretical support is provided by a series of calculations that describe a phenomenon wherein highly reactive metabolites, such as BPDE, follow a very specific

site-mediated reaction pathway instead of simply reacting with any available nucleophile

[23]. Various experiments have been cited as evidence involving electrophiles, PAH species in particular, that involve a non-covalent interaction prior to a substrate-ligand binding type reaction. Site-specific reactions such as that proposed for CHY-BPDE are typically explained by chemical reactivity, steric accessibility or active site proximity to nucleophilic residues, but no individual explanation has been broadly successful in describing why only certain residues are targeted, and why only those found in specific positions as opposed to that residue type within the entire protein.

A kinetic scheme for the reaction of small electrophiles with proteins can also be established. Assigning ka and k-a as the rate constants for reversible, noncovalent protein- electrophile binding, the ratio of ka to k-a then defines the equilibrium association constant. k1 is representative of the rate constant for reaction of an electrophile E with a surface-accessible nucleophile N1. It is assumed that k1 is not substantially different from the rate constant that characterizes the same reaction with a free amino acid or any other small molecule that has N1 as part of its structure. k2 is representative of the rate constant for covalent bond formation between E and nucleophile N2, which is located within the binding site for E. Some values for ka, k-a, and k1 have been measured or estimated, but values for k2 are largely unknown (Fig. 2-5) [23].

Figure 2-5. Representative diagram of potential equilibrium reactions between an electrophile E and neuleophilic residues N1 and N2 [23].

Equilibrium association constants are driven by association kinetics that are extremely

fast as compared to nucleophilic substitution reactions [23]. Implying a potentially high

kinetic favoring of the 2 step, non-covalent - covalent binding, over the one step,

unassisted reaction. To give further evidence of this case the work describes the

intercalation of Benzo[a]Pyrene-diol-epoxide (BPDE) with DNA occurring on a

5 - - millisecond timescale, with a calculated Keq = 10 M s for the BDPE-DNA association.

This suggests that even low affinity binding will probably exhibit rapid association kinetics, regardless of the strength of Keq.

Applying this theory of a “docking” based mechanism for this reaction type to a

albumin-BPDE adduct wherein a known reaction occurred in the 1B sub domain, a low

affinity-binding domain, gives further evidence of its plausibility. The adduct products

obtained were a His146 adduct with the (+) enantiomer and an Asp187 or Glu188 adduct with the (-) enantiomer. Upon determination of Albumins structure via x-ray

crystallography it was shown that these residues are in close proximity to each other and

the selectivity is understandable given that Albumin only exists as a single enantiomer

itself. This example elucidates the intercalation event that coordinates the PAH, leaving

only the front or back side reaction pending the specific BPDE enantiomer in question

[18].

Applying these theoretical calculations to CHYs histidine residue located in the center

of its catalytic triad associated with the presence of a hydrophobic pocket within the

receptor binding site, one specifically “designed” to accommodate the large hydrophobic

residues; tyrosine, tryptophan and phenylalanine, allows for a possibility of the CHY-

BPDE adduct formation. Additionally CHY only has 11 surficial nucleophiles, 7 aspartic

acid and 4 glutamic acid residues, giving the enzyme a low nucleophilic character, which

also implies a likelihood for receptor site mediated binding. Incorporating CHY’s small number of external nucleophiles and its hydrophobic pocket adjacent to the active site this theoretical work bolsters the hypothesis that the active site of Chymotrypsin makes a very “enticing” nucleophile for the BPDE specie.

2.2. Methodology

2.2.1. CHY adduct formation

2.2.1.1. CHY-TPCK adduct reaction

10 mg of protein was dissolved in 1 mL of 10 mM sodium/potassium phosphate buffered normal saline, pH 7.4. To this is added 100 µL of an approximately 1 mM solution of TPCK in 2:1 methanol (MeOH)/H2O. The reaction is allowed to proceed for 1

h at room temperature.

2.2.1.2. Proposed CHY-PAH adduct reaction

The BPDE-protein reaction is a modification of the methods described for the reaction

of ± (anti) r-7, t-8- dihydroxy- t-9, 10-epoxy-7, 8, 9, 10 tetrahydro Benzo[a]pyrene with

human hemoglobin, human serum albumin and bovine serum albumin [18].

100 mg of protein will be dissolved in 4mL of 10 mM sodium/potassium phosphate buffered normal saline, pH 7.4. To this is added 1 mg of the BaP diol epoxide in 100 µL

tetrahydrofuran (THF). The reaction is allowed to proceed for 2 h at 37 °C. The reaction

is terminated by extraction with ethyl acetate and the remaining solution is then washed 3

times with 4 mL aliquots of n-butanol. 10 mL diethyl ether is then added to the organic

phase extract and it is dried under a stream of N2 [18].

2.2.2. Whole protein adduct analyses

2.2.2.1. LC separation and QQQ analysis:

Separation was performed by high pressure liquid chromatography (HPLC) using a

reversed phase Vydac protein C-4 100 x 0.3 mm, 5 µm, 300A column. Solvent A consists of H2O with 1 % acetonitrile (ACN) and 0.1 % formic acid. Solvent B was an

ACN solution containing 0.1 % formic acid. 50 mg of the dried protein extract was

dissolved in 2 mL 50:50 H2O/ACN for evaluation. The column is pre-equilibrated with

10 % solvent B. Initial linear gradient is from 10 – 50 % in 30 min, followed by a 5 min

isocratic elution, followed by a second gradient segment from 50 – 85 % acetonitrile

solution in 10 min followed by a 15 min isocratic elution.

Average molecular masses were measured by triple quadrupole mass spectrometry to

determine adduct formation, and number of adducts/molecule if multiple adducts are

observed. Protein mass is determined using Mass Lynx software package. Settings for

whole protein were used, CHY samples were prepared and both infused directly in the

MS by controlled flow needle injection system or introduced by HPLC. Samples

introduced via HPLC injection were analyzed using the described gradient ramp. A

special apparatus was constructed to control back pressure and optimize LC conditions to

maintain post column flow of 45μL (Appendix B, Fig. B-1). The pump was set at

approximately 0.60 mL / min to maintain 55 psi on column. From these samples ESI

polarity, Cone Voltage, Capillary Ion Flow, HV lens, scan time and data range were

optimized on analysis for sample detection.

To verify LC QQQ MS retention time reproducibility control samples were analyzed

at the start and finish of assigned MS research days. Column and LC-MS conditions were evaluated using a tryptic myoglobin digest sample. Tryptic Digest Conditions: Scan

Range 280-1500, 45 cone voltage, 2.5 step, LC gradient 0-80 min 2-70% B, 80-90 min

70-90%B 110 min end of run.

2.2.2.2. LC separation and Ion Trap analysis:

HPLC ESI-Ion Trap MS was carried out by injecting the sample offline into a secondary LC loop using a Loading Phase of 2% ACN in H2O with 0.2% HCOOH and

collecting the molecules in a C-4 Cap Trap (MicromResources). The sample was washed for 10 min in the Cap Trap and then main column switch brings the CapTrap online into the primary Agilent LC mobile phase ramp; the column switch is diagramed in appendix

B, Fig. B-2.

Post isocratic cap-trap enrichment, samples were analyzed under a gradient ramp, as described above. LC flow conditions of 5µL / min to maintain a graded 75 – 30 psi on

column corresponding with net mobile phase polarity. From these samples ESI polarity,

cone voltage, capillary ion flow, HV lens, nebulizer psi, flow rate of dry gas, temperature,

ESI voltage, scan mode, range, speed, ICC Smart Target, maximum accumulation time

and scan range were optimized on analysis for sample detection.

As a control for LC ESI-Ion Trap the 66 kDa albumin marker from SDS-PAGE gel

samples was chosen. This control was used to optimize protein parameters and used for

all Ion Trap experiments.

2.2.3. Hydrazinolysis analysis

2.2.3.1. Hydrazinolysis

Ten mg of the dried protein extract was dissolved in 0.5 mL hydrazine and kept at 100

°C in a reflux condenser. After 15 h the solution was chilled in an ice bath. The solution

was then applied on water-flushed C-18-SPE column, prewashed with MeOH. The

column was then sequentially washed with 2 mL water and 2 mL MeOH/water (3:7).

The BPDE products were then eluted with 2 mL MeOH/water (8:2) and the solvent was

evaporated to dryness under a stream of N2 [23]. The sample was reconstituted in 100 µL

50 % ACN / 50 % H2O for injection.

2.2.3.2. LC separation and QQQ analysis

Separation of hydrazinolysis products was performed by HPLC using a reversed phase

Vydac C-18 column, 100 x 0.3 mm, 5 µm, 300A. Solvent A consists of H2O with 1%

ACN and 0.1 % formic acid. Solvent B was an ACN solution containing 0.1 % formic

acid. 10 µL of the peptide solution was injected for evaluation. The column was pre- equilibrated with 10 % acetonitrile solution. Initial linear gradient is from 10 – 40 %

acetonitrile solution in 30 min, followed by a 5 min isocratic elution, followed by a

second gradient segment from 40 – 60 % acetonitrile solution in 10 min and by a third

gradient to 90 % acetonitrile solution in 10 min, with 5 min of isocratic elution.

HPLC ESI-QQQ MS was carried out by splitting the outlet of the HPLC column in

two directions: one to the UV detector and the other to mass spectrometer with

electrospray ion source. A second apparatus was constructed to control back pressure on

column for sample injection with outlet split to maintain 45 µL / min post column flow

rate (shown in appendix B, Fig. B-3). UV detection was set at 345nm, specific to BPDE products. Average molecular masses measured by mass spectrometry was be used to

determine adduct formation and initial amino acid identity of the amino acid residue-

PAH adducts.

For optimization of experimental conditions and QQQ settings for small molecules,

histidine, B[a]P, a 7, 8 diol Benzo[a]pyrene(BPD), and TPCK solutions were prepared and infused directly by controlled flow needle injection system. Simple QQQ mass spectra for these samples can be seen in Fig. 2-6. From these samples ESI polarity, Cone

Voltage, Capillary Ion Flow, HV lens, scan time and data range were optimized for sample infusion. Parameters for collision induced dissociation (CID) and formation of daughter fragments were also optimized as seen in the infusion experiment of TPCK (Fig.

2-7).

Optimization of parameters for small molecules by sample introduction via LC injection was also performed for method development. Samples were injected under the gradient described at 0.625 mL /min, 48 psi to maintain post C-18 column flow of 45 μL.

For tandem MS analysis fractions were collected at 1 min intervals for subsequent infusion and CID daughter analysis.

2.2.3.3. LC Separation and Ion Trap analysis

Separation of hydrazinolysis product was performed by HPLC using a reversed phase

Vydac C-18 column, 100 x 0.3 mm, 5 µm, 300A. Solvent A consists of H2O with 1%

ACN and 0.1 % formic acid. Solvent B was an ACN solution containing 0.1 % formic

acid. 10 µL of the peptide solution is injected for evaluation. The column was pre-

Figure 2-6. QQQ Mass spectra of histidine and TPCK, optimization of small molecule analysis.

Figure 2-7. QQQ Mass spectra of TPCK and daughter analysis of parent molecule,

optimization of small molecule analysis CID.

equilibrated with 10 % acetonitrile solution. Initial linear gradient is from 10 – 40 % acetonitrile solution in 30 min, followed by a 5 min isocratic elution, followed by a second gradient segment from 40 – 60 % acetonitrile solution in 10 min and by a third gradient to 90 % acetonitrile solution in 10 min, with 5 min of isocratic elution.

HPLC ESI-Ion Trap MS was carried out by injecting the sample “offline” into a secondary LC loop using a Loading Phase of 2% ACN in H2O with 0.2 % HCOOH and collecting the molecules in a C-18 Cap Trap (MicromResources). The sample was washed for 10 minutes in the Cap Trap and then main column switch brings the CapTrap

“online” into the primary Agilent LC mobile phase ramp (appendix B, Fig. B-2).

For optimization of experimental conditions and Ion-Trap settings for small molecules histidine, B[a]P, a 7, 8 diol Benzo[a]pyrene(BPD), and TPCK solutions were prepared and introduced by HPLC. Direct infusion was not utilized for these samples to allow for additional sample enrichment and to remove any buffer prior to column and trap exposure. Post isocratic cap-trap enrichment, samples were analyzed under the described gradient ramp at a LC flow rate of 5µL / min to maintain < 75 psi on column. From these samples ESI polarity, cone voltage, capillary ion flow, HV lens, nebulizer psi, flow rate of dry gas, temperature, ESI voltage, scan mode, range, speed, ICC Smart Target, maximum accumulation time and scan range were optimized for sample detection.

2.2.4. Enzymatic digest analysis

Trypsin was obtained from Promega or Pierce. Protein digestion was performed on 1-

D SDS-PAGE gel. Gel bands of interest were excised, cut into approximately 1mm3 pieces and stored at -20°C until digestion. For digestion gel pieces were

washed/destained with 200 µL wash reagent (50% Et-OH / 5% acetic Acid / 45% H2O), for 1 hr. Wash was discarded and wash step repeated for an additional hour. Gels were then dehydrated by adding 200 µL acetonitrile for 10 min. Acetonitrile was discarded and samples were rehydrated in ammonium bicarbonate for 5 min. Ammonium bicarbonate was discarded and samples are again dehydrated with acetonitrile for 10 min.

Acetonitrile was discarded and the samples were dried in SpeedVac for approximately 3-

5 min. Samples are rehydrated in 50 µL trypsin, prepared to approximately 20 ng/µL and incubated at room temperature (23-25°C) overnight.

After digestion step samples were microfuged and the supernatant digest mixture collected. To the remaining gel pieces 30 µL extraction reagent was added (50 % ACN /

5 % formic acid / 45 % Water), sample was briefly vortexed and left for 10 min. The extraction mixture was removed and added to the digest mixture, the extraction step is repeated once. Combined supernatants were placed in SpeedVac to reduce volume to approximately 10 µL, about 45 min – 60 min. Sample volumes were reconstituted to approximately 25 µL total volume with 1 % acetic acid for LC-MS analysis.

HPLC ESI-Ion Trap MS was carried out by injecting the sample offline into a

secondary LC loop using a loading phase of 2% ACN in H2O with 0.2 % HCOOH, the

molecules are enriched on a C-18 CapTrap (MicromResources). The sample was washed

for 10 minutes in the CapTrap and then main column switch brings the CapTrap online

into the primary Agilent LC mobile phase ramp as described in 2.2.2.3. Ion Trap

conditions were set to those determined for small molecule analysis and optimized on analysis as needed.

2.2.5. Functional analysis of protein

The activity of chymotrypsin was based on the following reaction [24]:

N-Benzoyl-L-Tyrosine Ethyl Ester + H2O  N-Benzoyl-L-Tyrosine + Ethanol

The reaction velocity was determined by measuring an increase in absorbance at 256 nm resulting from the hydrolysis of benzoyl-L-tyrosine ethyl ester (BTEE). One unit hydrolyzes one micromole of BTEE per min at pH 7.8 and 25ºC under the specified conditions. The reaction was continuously observed in a spectrophotometer at 256 nm,

25º.

Ten mg of protein extract was dissolved in 10 mL 0.001 N HCl. The protein solution was then diluted with 0.001 N HCl to give 10-30 µg protein/mL. Into a cuvette the following reagents were added; 500 µL 0.08 M Tris HCl buffer, pH 7.8 with 0.1 M

CaCl2, 467 µL 0.00107 M BTEE in 50 % w/w methanol (63 mL absolute methanol

added to 50 mL Mili Q water). The reaction mixture was then incubated in the

spectrophotometer at 25ºC for 5 minutes to achieve temperature equilibrium. 33 µL of

the protein solution was then added and the absorbance at 256 nm is recorded for 20 min.

2.3. Results & Discussion

2.3.1. QQQ MS characterization of adduct in chymotrypsin

Preliminary model analysis settings for QQQ MS operation were established as

described. All analysis was conducted under positive polarity. Cone voltage settings

were modified and optimized for whole protein, peptide, or small molecule analysis as a

function of optimal scan range. Cone voltage was adjusted accordingly: average cone

voltage: 35 v = 200 m/z – 400 m/z, 40 v = 500m/z, 70 v = 600 m/z – 700 m/z, 90 v =

2000 m/z. A setting of 35 v for hydrazine digest, 70 v for tryptic digest and 75 v for whole protein analysis was selected. HV lens, scan time and data range were selected as a function of sample type, a shorter scan time and data range chosen for the whole protein analysis and a longer scan time with a wider data range for the peptide/small molecule analysis.

Whole protein samples of CHY and CHY-TPCK were analyzed by direct infusion and the LC C-4 methods described (Fig. 2-8). Hydrazine protein digests were then analyzed using the described gradient with LC C-18 separation to establish retention times and develop fractionation procedure. Comparative data of CHY and the CHY-TPCK digest chromatograms display high peak elution conservation with an interesting peak seen only in the adduct sample (Fig. 2-9) Of some concern in observing our adduct, was the potential cleavage of the TPCK-Histidine bond due to a Gabriel synthesis type reaction of the Hydrazine with the unknown reactivity of the imidazole bound nitrogen. This potential reaction could limit detection of the model adduct, and our proposed PAH adduct; based on current literature no documentation of TPCK-Histidine adduct has been characterized. However our successful observation of the TPCK-Histidine product has verified that any additional reaction by hydrazinolysis, if it occurs, is not complete under these conditions.

Theoretical amino-acid hydrazide molecular weights were calculated (Table 2-I) and compared to QQQ-MS results. The peak observed at 42.6 min in the CHY-TPCK digest not seen in the CHY digest was collected by fractionation and further analyzed by infusion QQQ MS (Fig 2-10) fitting a potential match to the His-TPCK hydrazide.

Figure 2-8. LC-MS QQQ Whole CHY-TPCK (top) and CHY (bottom).

Figure 2-9. LC MS QQQ Separation of hydrazine digests of CHY-TPCK (top) and

CHY (bottom).

Figure 2-10. LC MS QQQ hydrazinolysis peak spectra at 42.6 min in CHY-TPCK digest corresponding to Histidine – Adduct.

CGVPAIQPV……IVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAH

CGVTTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAAS

FSQTVSAVCLPSASDDFAAGTTCVTTGWGLTRY..ANTPDRLQQASLPLLSNTNC

KKYWGTKIKDAMICAGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCS

TSTPGVYARVTALVNWVQQTLAAN

-Underlines refer to Sheet segments and Italics to Helix segment

Residue # in CHY Monoisotopic Mass AA-Hydrazide Mass Glycine (G) 22 57.02146 89 Alanine (A) 22 71.03711 104 Valine (V) 23 99.06841 131 Leucine (L) 17 113.08406 145 Isoleucine (I) 10 113.08406 145 Proline (P) 9 97.05276 130 Phenylalanine (F) 6 147.06841 179 Tyrosine (Y) 4 163.06333 195 Tryptophan (W) 8 186.07931 219 Cysteine (C) 10 103.00919 135 Methionine (M) 2 131.04049 163 Serine (S) 26 87.03203 119 Threonine (T) 22 101.04768 133 Lysine (K) 14 128.09496 160/161+ Arginine (R) 3 156.10111 188/189+ Histidine (H) 2 137.05891 169/170+ Asparagine (N) 13 114.04293 146 Glutamine (Q) 10 128.05858 160 Aspartic Acid (D) 9 115.02694 147 Glutamic Acid (E) 5 129.04259 161

Table 2-I. Secondary structure of chymotrypsin, individual amino acids molecular

weight and the molecular weight of the expected amino acid hydrazides.

LC parameters and MS-QQQ settings were continuously monitored to optimize data

collection. Key focus was placed on peak resolution and tR reproducibility for later fraction collection. These continued modifications of digest protocol and refinement of

LC-MS parameters allowed for a large amount of accurate data on the digest system to be acquired.. The drawback to this system is that initial data is only MS1 as the QQQ-MS

lacks the specificity to accurately verify compounds without fractionation and subsequent

infusion. Arguably samples could be analyzed in repetition and a precisely programmed

protocol to alter MS* settings for each peak of interest could be established. This

additional procedure would then be required for each system being observed, presenting

the immediate drawback of large sample volume and long MS analysis times. At this

point the project was moved to the departments newly acquired the Ion-trap MS and the

work on the QQQ was re-focused for the development of ESI-Ion Trap experimentation

that would allow for MS* analysis of compounds directly in-line with the initial

identification step.

2.3.2. LC ESI-MS Ion characterization of adduct in chymotrypsin

Whole protein samples of CHY and CHY-TPCK were analyzed by the LC C-4

methods described. Hydrazine protein digests of CHY and CHY-TPCK were then

analyzed using the described gradient with offline captrap washing and enrichment

followed by C-18 separation and MS. Comparative data of CHY and the CHY-TPCK are

shown in (Fig. 2-11). MS2 spectra were acquired for peaks and a potential Histidine-

TPCK hydrazide peak was observed at a similar retention time, 46 min (Fig. 2-12).

Figure 2-11. LC MS Ion Trap Separation of hydrazine digests of CHY-TPCK (top) and CHY (bottom).

Figure 2-12. LC MS Ion Trap hydrazinolysis peak spectra at 46.4 min in CHY-

TPCK digest corresponding to Histidine – Adduct.

2.3.3. UV Spectroscopic Analysis

For optimal activity CHY was prepared in a Ph 7.8 Tris buffer system. Reaction

kinetics were evaluated using UV-Spec of various concentration TPCK – CHY solutions

and allowed varied exposure times prior to analysis for determination of optimal inhibition. Enzyme activity was reduced substantially after 30 min allowing an effective

and fast means of determining loss of proteolytic activity in CHY (Appendix B, Fig. B-

4).

2.3.4. Tryptic Digest

As further development of mass spectrometric methods of analysis and sample preparation for study was employed an additional means of enzyme digestion was explored. With a wealth of bioinformatics data available a tryptic digest would prove very useful in protein – adduct experiments and was primarily pursued here as a model method for potential future studies.

Trypsin, a serine protease, is a digestive enzyme that specifically cleaves polypeptides on the carboxylic side of lysine and arginine residues. Native trypsin is subject to

autolysis, a self cleaving process that can produce pseudo-enzymes that exhibit a wider

range of cleavage sites, including chymotrypsin like activity. Trypsin Gold is

manufactured porcine trypsin in which the lysine residues have been modified by reductive methylation, resulting in a highly stable enzyme. Additionally the prepared trypsin is treated with TPCK to inhibit any chymotrypsin activity and then purified further by affinity chromatography and lyophized.

CHY was digested both in solution and via SDS-PAGE Gel isolation with Promega

mass spectrometry grade trypsin gold. In this experiment the tryptic digest of CHY did

not yield conclusive peptide-adduct data, most likely as a result of CHY and trypsin’s

inherently bound functional roles.

2.4. Conclusions

Developing an efficient and effective means of evaluating PAH-protein adducts is an important step in understanding the full effect these pollutants may have on cellular function and biochemical pathways. The ubiquitous nature of these pollutants alone should interest all sectors of society in the determination of the immediate effect of toxin exposure on proteins in terms of both structural modification and proteolytic function.

This study positively identified an adduct event on the whole protein by loss of enzymatic activity and by expected mass shift, also by chemical degradation of the protein to its constituent amino acids and detection of an adduct-hydrazide. The reaction

with hydrazine allows the determination of adduction to a specific residue type, but offers

no additional information as to where on the proteins structure the adduct may be.

Utilizing an enzymatic digestion of a protein could offer additional insight into the adduct formation, and possibly identify the specific sight of adduction. Upon tryptic digestion of the protein the resulting peptides are well matched to ESI ion trap analysis by nature of their N and C termini. CID analysis of the peptides within the trap would allow for further sequence information of the peptides secondary structure. Taking this a step further one can incorporate knowledge of a proposed adduct reaction type and write

a modification specific search protocol for the available bioinformatics database. The

data then obtained from the MS can be searched against the expected tryptic peptides for the protein while allowing for the potential mass shift of the adduct. In whole this would offer a much greater confirmation than the chemical degradation alone.

Future steps in this project call for an exposure of CHY to BPDE. CHY function can be reliable studied by enzymatic assay and whole protein and amino acid modification determined by LC ESI ion trap MS. Additional research direction would involve the incorporation of tryptic digestion of proteins of interest upon exposure to PAHs for more accurate determination of adduction sites.

2.5. References

1. Environmental Health Criteria 202, IPCS, World Health Organization:

Geneva, 1998; 47- 96.

2. Cerniglia C.E. (1992) Biodegradation of polycyclic aromatic hydrocarbons.

Biodegradation 3:351- 368.

3. Pavanello, S., Clonfero, E. (2000) Biological indicators of genotoxic risk and

metabolic polymorphisms. Mutation Research 463:285 – 308.

4. Angerer, J., Mannschreck, C., Gundel, J. (1997) Biological monitoring and

biochemical effect monitoring of exposure to polycyclic aromatic

hydrocarbons. Int Arch Occup Environ Health 70:365-377.

5. Hemminki K., et al. (1990) DNA adducts in humans related to occupational

and environmental exposure to aromatic compounds. In: Vainio H, Sorsa M,

McMichael AJ (eds) Complex mixtures and cancer risk. IARC, Lyon pp181-

192.

6. Ovrebo S, et al. Biological monitoring of exposure to polycyclic aromatic

hydrocarbon in an electrode paste plant. J Occup Med 36:303 – 310.

7. Phillips D H, et al. (1998) Mutat Res 204:5331-541.

8. Reddy M V, Hemminiki K, Randerath K (1991) Post labeling analysis of

polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of

foundry workers. J Toxicol Environ Health 34:177-185.

9. Petera F P, et al. (1994) Carcinogen-DNA adducts and gene mutation in

foundry workers with low-level exposure to polycyclic aromatic

hydrocarbons. Carcinogenesis 15:2905-2910.

10. Hemminiki K, et al (1990) Aromatic DNA adducts in white blood cells of

coke workers. Int Arch Occup Environ Health 62:467 – 470.

11. Ovrebo S, et al (1995) Studies of biomarkers in aluminum workers

occupationally exposed to polycyclic aromatic hydrocarbons. Cancer Detect

Prev 19:258-267.

12. Herbert R, et al. (1990) detection of adducts of DNA in white blood cells of

roofers by 32P post labeling. Scand J Work Environ Health 16:135-143.

13. Santella R M, et al. (1995) Polycyclic aromatic hydrocarbon-DNA and protein

adducts in coal tar treated patients and controls and their relationship to

glutathione S-transferase genotype. Mutat Res 334: 117-124.

14. Kure E H, Andreassen A, Ovrebo S, et al. (1997) BaP-hSA adducts in humans

exposed to PAHs in an industrialized area of Poland. Nor – Occup Environ

Med 54/9:662- 666.

15. Willett K L, et al. (1998) Inhibition of CYP1A1- Dependant Activity by the

Polynuclear Aromatic Hydrocarbon Flouranthene. Biochem Pharm 55: 831-

839.

16. Brunmark P, et al (1998) Acta Chem Scand 52:797.

17. Ferranti P, et al. (1996) Carcinogenesis 17:2661.

18. Helleberg H, and Tornqvist M. A new approach for measuring protein

adducts from BaP diolepoxide by HPLC/MS, Rapid Communication in Mass

Spectrometry. 14:1644-1653

19. Mraz J, et al 6th European Meeting on Mass Spectrometry in Occupational

and Environmental Health, September 1-3 1999, Stockholm, Scientific

Program and Book of Abstracts, Ostman C (ed). Acro Media, Stockholm

University: Stockholm, 1999; 70-71

20. Sabbioni G., et al. (2001) Chem Res Toxicol. 13:82.

21. Brunmark P., Harriman S., Skipper P.L., Wishnok J.S., Amin S., Tannenbaum

S.R. (1997) Chem. Res. Toxicol. 10:880.

22. Day B.W., Skipper, P.L., Zaia J., and Tannenbaum S.R. (1991)

Benzo[a]pyrene anti-diol epoxides covalently modifies human serum albumin

carboxylate side chains and imidiazole side chain of histidine (146). J. Am.

Chem. Soc. 113:8505–8509.

23. Skipper, Paul L. (1996) Influence of Tertiary Structure on Nucleophilic

Substitution Reactions of Proteins. Chem. Res. Toxicol. 9: 918–923.

24. Hummel, B. (1959) A Modified Spectrophotometric Determination of

Chymotrypsin, Trypsin and Thrombin. Can. J. Biochem. Physiol. 37:1393

CHAPTER III

MS DETERMINATION OF POLYCYCLIC AROMATIC

HYDROCARBONS (PAHS) IN SEDIMENT AND WATER

SAMPLES FROM BOLGODA AND BEIRA LAKES

3.1. Abstract

Eleven polycyclic aromatic hydrocarbons (PAHs) have been quantified in surface sediment and aqueous samples from the Bolgoda and Beira lakes, Sri Lanka. This study represents an initial attempt to study the potential impacts of PAHs on the lakes. Water and sediment samples were collected from four sites in Beira Lake which is situated in downtown Colombo, capital city of Sri Lanka, and from six sites in Bolgoda Lake which is located on an outskirt of Colombo but is surrounded by a number of busy townships dotted with a variety of small industries. The lake also receives massive amounts of industrial effluents from an industrial center located upstream. Total concentrations (sum of 11 PAHs) of PAHs among the four sediment samples in the

Beira Lake showed a wide site to site variability ranging from 151 to 568 µg/kg, while

the concentration range in the surface water was 46 – 84 ng/L. The PAH concentrations

in the sediments in Bolgoda Lake ranged from 115 to 934 µg/kg, and their level in

water samples was between 40 and 127 ng/L. In general the PAH composition in the

sediments varied from that in the water column. The total PAH concentrations in the

sediments appeared to reflect the proximity of the sampling site to anthropogenic sources.

The PAHs of Beira Lake sediments and waters were dominated by those with 4-5 rings:

66 – 75% in water and 71 – 92% in sediments. Similar concentration pattern was

observed for the Bolgoda Lake: 57 – 61% for water and 71-89% for sediments, a strong

indication of pyrolytic inputs. The applications of anthracene/phenanthrene and

fluoranthene/pyrene ratios also indicated dominant pyrolytic inputs. In addition, the

presence of high 1-nitropyrene levels at several sites indicates a strong contribution from

mobile sources; especially from diesel-powered vehicles. Short-term solid-phase

Microtox assays showed toxicity of the sediments at all sites; however there was little correlation between the results of the assay and total PAH concentrations.

100

3.2. Introduction

The pollution of polycyclic aromatic hydrocarbons (PAHs) has been widely used to

assess the potential impact of anthropogenic activities on aquatic environments

because their occurrence in water is closely tied to urban activities. Many PAHs

possess mutagenic and carcinogenic properties [1,2]. PAH distribution and toxic

potentials therefore have been the focus of numerous studies in waterways including

the Great Lakes [3], Yanisei Bay [4], and the Fraser River basin [5]. Sri Lanka, a small island nation with a dense population of about 20 million people, faces a multitude of environmental stresses ranging from deforestation to traffic congestion and the deterioration of water quality. This study was undertaken to understand the occurrence, sources, and potential impacts of PAH in the waterways in Sri Lanka. Two lakes, Beira and Bolgoda lakes, were selected for the study due to their economic value and high level of pollution. Beira Lake, situated in downtown Colombo, the capital city of Sri Lanka, is highly polluted. Sources of pollution are multivarious. For instance, clusters of

communities have sprung up along the edges of the lake in recent times and many shacks

have been built. These communities are generally not connected to municipal sewer

systems and substantial quantities of domestic sewage and untreated wastewaters are

discharged directly into the lake. Small industries have also grown rapidly around the

lakes, most of which are not believed to have adequate facilities to treat industrial

wastewater, especially organic wastes. In addition, Sri Lanka has experienced an upsurge

of motor vehicles, including millions of three-wheelers and minivans that are powered by

leaded-gasoline and diesel fuels. Traffic congestion and severe air pollution due to

vehicle emissions now is a common daily occurrences and is considered major potential

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sources of PAH in the lakes. Although Bolgoda Lake is situated some distance from

Colombo, it is heavily polluted due to the growing number of towns with attendant

increase in small businesses and various industries along its shores. These new developments have undoubtedly impacted the lake through the discharge of PAHs and

other anthropogenic chemicals present in industrial wastewater and from street runoffs.

The lake, additionally, receives a large quantity of pollutants from the industrial zone in the north.

The pollution caused by PAH has led to various studies on the distribution and origin of PAHs in environment [6,7]. Based on the proportions of different PAH, most studies aim to distinguish PAHs of petrogenic sources from those of pyrolytic origins. The

PAHs of petrogenic origin, prevalent in coals and fossil fuels, are formed from diagenesis of sedimentary organic material under low to moderate temperature and tend to consist of low molecular weight PAHs with two to three aromatic rings [8]. The pyrolytic PAHs, on the other hand, are formed at much higher temperatures, for example, greater than

500oC, and consist mainly of four and higher number of rings [9]. Thus, an increase in the

proportion of higher molecular weight PAHs is interpreted to indicate contamination of

mainly of pyrolytic origin. The prevalence of high molecular weight PAHs in the urban

dusts [10] and in atmospheric particles [11] illustrates the chemistry of their formation at

high temperature.

For this study 11 known potential toxinogenic and mutagenic PAHs [12,13] were

chosen: naphthalene, acenaphthene, phenanthrene, anthracene, benzo(k)fluoranthene,

fluoranthene, pyrene, chrysene, 1-nitropyrene, benzo(a)pyrene, and

dibenz(a,h)anthracene. Of these PAHs, five have 4-5 aromatic rings

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(benzo[k]fluoranthene, pyrene, chrysene, benzo[a]pyrene, and dibenz[a,h]anthracene) and the remaining four have 2-3 rings (naphthalene, acenaphthene, phenanthrene, and anthracene).

3.3. Methodology

3.3.1. Sample Site Selection & Collection

Fig. 3-1 shows a map of Beira and Bolgoda Lakes, indicating the various sampling sites. In comparison to most lakes around the world, both lakes are characterized by extreme shallowness with an average depth of 2–3 m, and being in a tropical region near the equator, they experience little seasonal variation of water temperature (26-28oC) and receive frequent rainstorms, ranging from local thunderstorms that can occur almost daily to the southwest monsoons from May to September. In the catchment area where the lakes are located, rainfall ranges from about 4 inches in January to nearly 14 inches in

October.

Beira Lake is situated well within the city limit of Colombo and opens to the Indian

Ocean through a short and narrow canal in the west, and extends eastward by branching into West and East Lakes. The West Lake narrows to a strip of water and meanders into another small lake, S.W. Lake. The exchange of water between these lakes is very slow. Fresh water flows into the eastern and northern parts of the lake through a number of small tributaries. Three sampling sites BA-1 and BA-3, and B-4 in

Beira Lake, are in the vicinity where reclamation work was being performed. These areas are surrounded by a high density of shops and busy streets, and a large number of

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people have built shacks along the edges of the lake. Site BA-2 is in a region where the

shops are not as numerous and the traffic is less congested.

Bolgoda Lake is also located in the western Sri Lanka and opens to the Indian Ocean

via the two narrow, winding canals. Fresh water flows into the lake mainly by the Weras

Ganga from the Dehiwala area and the eastern drain of Lady Catherine industrial region.

In addition, the lake is surrounded by a substantial number of small shops and industries,

some of which are considered as potential sources of PAHs. Sampling sites WG-1 and

WG-2 are located in the mouth of North Lake and BNL-1 is in the middle of North Lake.

These sites are therefore susceptible to being impacted by the water pollutants and

contaminants originating from the industrial zone in the north. BNL-2 is close to the

canal where the exchange of water between the ocean and lake occurs. Site BG is away from the river Ganga, hence the water quality is expected to be more reflective of the immediate surroundings. Site BSL is along the canal area close to Bolgoda south and is

distant from the other study sites.

Surface sediment samples (0-3 cm) were collected using a scoop in the shallow point

of selected sites during 2003-2004. Water samples were also collected from the surface

in 1 L amber glass bottles with screw caps. Samples, collected in glass jars were stored

frozen at -20oC until analysis. Both colloid and surface sediment samples have been

collected in duplicate from selected sites at various times of the year.

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3.3.2. Sample Preparation

Moist sediments were subjected to Soxhlet extraction. The methodology for the

extraction and fractionation is similar to that described [14]. Briefly, 10 g sediments

were extracted with 50 mL of dichloromethane at three cycles per hour for 24 hours.

After cooling to ambient temperature, residual water was removed by adding 2-3 g of sodium sulfate. Then, the contents were evaporated at 35oC to near dryness (1-2 mL) and

to dryness under a gentle stream of nitrogen. The PAH fraction was enriched using

sephadex and silica gel chromatography in the following manner: the fraction is mixed

with a slurry of activated silica gel in dichloromethane and transferred to a small glass

column (10.0 cm x 1.0 cm id). The gel was equilibrated with 30 mL of pentane, 2 mL of

the extract was loaded, 25 mL of pentane was passed through, and the PAH fraction was

eluted with 25 mL of dichloromethane:pentane (4:6, v/v) and was taken to dryness

under a gentle stream of nitrogen. For analysis by high performance liquid

chromatography (HPLC), the extract was exchanged into acetonitrile. PAH recoveries

were determined by adding known quantities of PAH mixtures to the sediments (5-10

grams) and they were subjected to the same extraction procedures.

The water samples containing both dissolved and colloid-adsorbed PAHs were extracted

using a separatory funnel. Into a 1 L separatory funnel, 500 mL of water sample and 50

mL of CH2Cl2 were added and extracted for three times. PAH recoveries were checked by adding known quantities of PAH mixtures to distilled water. These spiked waters

were then extracted using the same procedures described in the previous paragraph.

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3.3.3. HPLC Sample Analysis

PAHs were analyzed by high performance liquid chromatography (HPLC) and gas chromatography coupled to mass spectrometry (GC-MS). The HPLC procedure was based on the U.S. Environmental Protection Agency (U.S. EPA) Method 8310 using high performance liquid chromatograph (Agilent 1100) with a gradient pumping system and a

250 mm by 2.6 reverse phase HC-ODS Sil-X 5-micron particle size column. A UV detector with excitation wavelength 254 nm was coupled to the fluorescence detector.

The fluorescence detector uses an excitation wavelength of 280 nm and emission greater than 289 nm cutoff. Quantitation was performed by the external standard method with individual PAH standards.

3.3.4. GCMS Sample Analysis

The GC/MS methodology was based on U.S. EPA 8100 Method. Samples were quantified by gas chromatography/mass spectrometry (GC/MS) on a Thermo Electron

GCQ plus ion trap mass spectrometer. Spiked samples containing a mixture of 11 PAHs and two reference compounds were analyzed for every 10 samples. Perfluoro-n- butylamine was used as the calibration compound to ensure instrument performance.

Samples were separated and analyzed on an Altech DB-5MS capillary column (0.32 mm

I.D. x 0.1 mm film thickness x 30 m). A Thermo Electron HS 2000 autosampler was used for all sample injections. Helium was used as carrier gas at a constant flow rate of 1.4 mL/min. The gas chromatograph oven temperature was programmed as follows: the initial temperature was maintained at 100oC and held for 1.0 min following injection. The temperature was then ramped at 20o C/min to 200o C and held there for 10 min. Finally

106

the temperature was ramped at a rate of 10o C/min to 300o C. The injection volume was 5

µL and the injector was maintained at 250o C. A splitless injection was used with septum purge operative throughout the analysis. The mass spectrometer was operated in Electron

Impact mode for all analysis with an electron energy of 70 eV, scanning at a mass range of 50 – 400 amu every 0.8 s. A five point calibration routine was used for all analysis of each PAH with at least one calibration point of higher or lower concentration than the concentration of the unknown compound under consideration. Peak areas were used to determine the response factors of each of the parent PAHs GC/MS retention times. The identity of PAHs was accomplished by comparison of spectral pattern of ions obtained to the libraries of mass spectral data. Perdeuterated aromatic compounds were used as internal standards for quantitative analyses in each aromatic series.

3.3.5. Evaluation of Sediment Toxicity

Vibrio fischeri NRRL B-11177 was obtained from Azur Environmental, Carlsbad,

CA, USA). The organisms were received freeze-dried and stored below – 20o C. Cells of V. fischeri were diluted to an absorbance of 0.4 at 650 nm in a simple medium that contained KH2PO4 (18.4 mM), NaCl (0.5 M), and MgSO4 (4.1 mM), The medium was adjusted to pH 7.2 and autoclaved prior to use

The solid phase Microtox assay was conducted as recommended by the manufacturers. The procedure involved placing a 1-g dry sediment sample in 5 mL assay medium in 10 mL borosilicate tubes and was used to make a series of sediment dilutions.

The sediment samples were serially diluted and 2 mL aliquots of evenly suspended samples were mixed with equal volume of test culture in polystyrene dishes (35 x 10

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mm). After 15 min of gentle shaking on a platform linear shaker at room temperature, the

contents were transferred to a polystyrene cuvette (12 x 75 mm) and luminescence was

measured in a tube luminometer (Zylux FB15). Luminescence in the assay medium

served as control. Phenol was used as reference toxin and EC50 values were maintained

within 13-26 mg phenol/L as recommended by the manufacturer and the luminescence

change of the test organism in the medium without the sediment served as positive

control.

Calculation of EC50 values were obtained from a plot of cumulative response (0-

100%) versus log sediment amount. Inhibition of luminescence intensity in the short-term test, for instance, was based on the following formula (McConkey et al 1997) = 100 [1-

(LfCi/LiCf)], where Li is the initial luminescence prior to and Lf is the luminescence following a 15-min exposure to sediment, and Ci and Cf are the initial and final luminescence of the bacteria that were not exposed to the sediment. The data for % light inhibition = 100/1 + e-β(lnx-lnµ), where x is the amount of sediment, µ is the EC50, and β is

the slope of the amount-response curve.

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3.4. Results & Discussion

3.4.1. Beira Lake

As shown in the map (Fig. 3-1 A) sampling in Beira Lake was conducted at four sites over a period of 18 months. Composite sediment and overlying water samples were collected from the sites and were analyzed for eleven parent PAHs. The overall concentration range for the sediments was found to be between 152 and 569 µg/kg with average 329 µg/kg dry sediment (Table 3-I). When the individual sites were compared, the total PAH concentrations from the BA-2 site were substantially below those obtained from the other sites (BA-1, BA-3, and BA-4). These sites are adjacent to an area of high traffic density, with numerous shops and small industries of various kinds in close proximity, thus direct local inputs of PAHs from the street runoff into the lake is probably most significant. Parts of the region around these sampling sites had recently undergone sediment dredging (hatched area) and are speculated to be heavily contaminated by metals and other contaminants. The region around the BA-2 site by comparison is less populated and the traffic not as congested.

Figure 3-1. Sampling sites in Beira Lake (A) and Bolgoda Lake (B).

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Table 3-I. Average PAH levels in Beira and Bolgoda Lakes a,b. ______

A. Beira Lake Sampling Sites

BA-1 BA-2 BA-3 BA-4______

Water (ng/L)

78 ± 5.0 46 ± 3.0 112 ± 4.7 85 ± 5.6

Sediment (µg/Kg)

569 ± 33.0 152 ± 8.1 465 ± 25.0 371 ± 21.0 ______

B. Bolgoda Lake Sampling Sites

WG-1 WG-2 BNL-1 BNL-2 BSL _____ BG__

Water (ng/L)

40 ± 2.2 127 ± 6.3 70 ± 3.7 67 ± 2.5 71 ± 2.6 71 ± 3.1

Sediment (µg/Kg)

406 ± 24.1 865 ± 50.1 115 ± 6.2 934 ±24.0 121 ± 6.8 249 ± 13.0

a Average of 11 PAH species: naphthalene, acenaphthene, phenanthrene, Anthracene, benzo[k]fluoranthene, fluoranthene, pyrene, chrysene, 1-Nitropyrene, benzo[o]pyrene, dibenz [a,h] anthracene b The mean ±SEM (N = 3 - 5)

The results from the analysis of overlying water samples in Beira Lake (Table 3-I A)

show that PAH concentrations in the water column at the BA-2 site were also lower

(46 ± 3.0 ng/L) than those of other three sites, which ranged 78 – 85 ng/L. Thus, there

was some correspondence between the aqueous and sediment samples in the PAH

concentrations at this site. For the most part, however, there were low correlations in the

concentration of PAHs between the phases. A comparison of individual PAHs also

110

indicated that PAH concentrations in the sediments did not reflect those of water column

(Fig. 3-2), probably due to a rapid mixing and transport of the PAH-adsorbed colloidal particles in shallow water column and that the majority of PAHs in water column are expected to be associated with colloidal particles since PAHs have low aqueous solubility as indicated by their high log Kow values (3.36 for naphthalene to

6.90 for coronene) [15]. The reduced site-to-site variability of the PAH concentrations in the water column could also be explained by the rapid equilibration of the colloidal particles. Various external factors such as wind velocity over the evaporating surface, frequency and intensity of rain storming events around the lakes, the effects of intense sunlight on the PAH degradation, and the relationship between year-round warm water temperature probably influence the loading of PAH in the lake.

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Figure 3-2. Relative distributions of PAHs for four sites in the water column and sediments in Beira Lake.

Values are mean ± standard error of mean. np=naphthalene, ac=acenaphthene, ph=phenanthracene, at=anthracene, bf=benzo(k)fluoranthene, f=fluoranthene, p=pyrene, ch=chrysene, np=1-Nitropyrene, bp=benzo(a)pyrene, dba=dibenzo(a,h)anthracene.

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3.4.2. Bolgoda Lake

Bolgoda Lake (Fig. 3-1 B), like Beira Lake, is also located in the catchment area but

some distance away from the city of Colombo. However, it is expected to experience a

substantial PAH loading since the lake is surrounded by a fair number of small industries,

such as incinerators and electricity power plants that are situated close to the lake, many of which produce gaseous emissions and generate wastewaters that are expected to contain high concentrations of PAHs. The PAH levels for the sediments ranged from

121 ± µg/kg at the BNL-1 to 934 ± µg/kg sediment at the BNL-2 site with the mean of

448 µg/kg sediment (Fig. 3-3). Overall PAH levels in sediment samples in the Bolgoda

Lake thus were somewhat higher than those of Beira Lake. The concentrations at WG-2

site were also high at 865 µg/kg. The potential sources of PAHs around the site are 16

small industries including a number of gas stations and a chemical industry, most of

which have the potential of discharging significant quantities of PAHs through

wastewaters as well as gaseous emissions. In addition, Bolgoda Lake receives massive

amounts of industrial effluents via the Ganga river, a major tributary, flowing out of the

Lady Catherine industrial zone in the north (Dehiwala area), and that the region around

site WG-2 was probably impacted by the effluents from the industrial zone more than the

other sites because of its location and other geologic characteristics. On the other hand,

heavy contamination of the sediments in BNL-2 site in the west was probably

attributed to the Panandura estuary where the exchange of lake water with the polluted

sea coast occurs and possibly reflects localized geographic variation of some unknown

origin. The extent of contribution of the PAHs present in the industrial effluents from

the industrial zone in Dehiwala area is unlikely to be important at this site because of

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the low levels of PAHs observed around the site BNL-1, which is closer to the WG-2

site. Located in the general vicinity of BNL-1 site are number of small industries such as a garment factory, several gas stations, and a large farm. Also, the general area is not as congested and the population is somewhat less than the areas in the vicinity of other sites.

These conditions perhaps explain the low levels of PAHs observed around the site. The

PAH levels for the remaining sites (WG-1, BSL, and BG) were also low, ranging between 115 – 247 µg/Kg. BG and BSL sites have one to two gasoline stations each.

None of the small industries in the area (for instance, hotels and shops) support activities expected to be directly associated with generation of PAHs. The total PAH concentrations for the six aqueous samples ranged from 40 to 127 ng/L with the average of 79 ng/L (Table 3-I B). The PAH distribution in the water column thus was similar to that of Beira Lake in that the PAH concentrations in the water column showed less variability for similar reasons.

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Figure 3-3. PAH Relative distributions of PAHs for six sites in the water column and sediments in Bolgoda Lake.

Values are mean ± standard error of mean. np=naphthalene, ac=acenaphthene, ph=phenanthrene, at=anthracene, bf=benzo( k)fluoranthene, f=fluoranthene, p= pyrene, ch=chrysene, np=1-Nitropyrene, bp=benzo(a)pyrene, dba=dibenzo(a,h)anthracene.

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3.4.3. PAH Ratios

An objective of this study was to determine potential sources of PAHs at each site.

Total concentrations of four- to five-ring PAHs were compared to those of two- to three

ring PAHs. Of the eleven major parent PAHs analyzed, five had 4-5 aromatic rings

(benzo[k]fluoranthene, pyrene, chrysene, benzo[a]pyrene, and dibenz[a,h]anthracene)

and the remaining four PAHs had 2-3 rings (naphthalene, acenaphthene, phenanthrene,

and anthracene). In Beira Lake the inputs of the PAH with 4-5 rings ranged from 58 to

66% of total concentrations for the water samples and 71 to 92% for the sediments (Table

3-II). A similar profile existed for Bolgoda Lake, where the PAHs with 4-5 rings comprised 57 – 62% for the water samples and 62-86% in the sediments. Thus, the distribution patterns of PAH in both lakes are indicative of dominant contributions from the pyrolytic sources at all sites, although significant site-to-site variation existed.

Alternatively, concentration ratios of phenanthrene/anthracene (P/A) and fluoranthene/pyrene (F/P) have been applied to distinguish between PAHs of pyrogenic and petrogenic sources [16-18]. This approach is based on the rationale that for parent

PAHs, combustion processes under the conditions of limited oxygen availability tend to produce proportionately more PAH isomers with greater thermodynamic stability [19].

For example, for the three ring PAHs, the proportion of more stable phenanthrene is expected to be produced in higher proportion than its isomer, anthracene, during the combustion processes. One report presented showed that PAHs of pyrolytic origin generally have P/A ratios of 1-10 [7]. The study further showed that fluoranthene/pyrene (F/P) ratios fall between 1-2 for the pyrolytic PAHs. The calculated values in Beira and Bolgoda lakes generally indicated dominance of pyrogenic sources

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Table 3-II. Ratiosa of two-three ringb to four-five ringc PAHs for Water and Sediment samples from Beira and Bolgoda Lakesd ______A. Beira Lake Sampling Sites BA-1 BA-2 BA-3 BA-4______Water (ng/L)

2-3 ring PAH 38 ± 2.0 38 ± 2.0 62 ± 4.0` 56 ± 3.4 4-5 ring PAH 112 ± 7.3 52 ± 3.5 154 ±5.2 108 ± 7.3 Ratio 75% 57% 71% 66%

Sediment (µg/Kg)

2-3 ring PAH 84 ± 4.3 82 ± 4.8 262 ± 12.5 100 ± 5.5 4-5 ring PAH 973 ± 56.9 210 ± 10.9 635 ± 35.5 597 ± 33.8 Ratio 92% 72% 71% 86% ______B. Bolgoda Lake Sampling Sites WG-1 WG-2 BNL-1 BNL-2 BSL BG Water (ng/L)

2-3 ring PAH 34 ± 2.3 74 ± 3.5 61 ± 3.2 51 ± 2.3 60 ± 2.9 53 ± 2.6

4-5 ring PAH 45 ± 2.1 171 ± 8.6 78 ± 4.2 81 ± 2.7 80 ± 2.4 86 ± 3.5

Ratio 57% 70% 56% 61% 57% 62%

Sediment (µg/Kg)

2-3 ring PAH 83 ± 4.4 377 ± 22.8 65 ± 3.4 360 ± 18.5 88 ± 4.7 67 ± 3.8

4-5 ring PAH 674 ± 40.6 1273 ± 72.9 156 ± 8.5 1413 ± 28.5 149 ± 8.5 400 ± 20.6

Ratio 89% 77% 71% 80% 63% 86%

a Average 4-5 ring PAH / Average 2-3 ring PAH x 100 b Average of 5 PAH species: naphthalene, acenaphthene, phenanthrene, anthracene, fluoranthene c Average of 6 PAH species: benzo[k]fluoranthene, pyrene, chrysene, 1-Nitropyrene, benzo[o]pyrene, dibenz [a,h] Anthracene d The mean ±SEM (N = 3 – 5)

117

Table 3-III. Phenanthrene/anthracene (P/A) and fluoranthene/pyrene (F/P) ratios for water and sediment samples from Beira and Bolgoda Lakes. ______A. Beira Lake Sampling sites______

BA-1 BA-2 BA-3 BA-4

Water P/A 0.9 3.6 3.0 14.5 F/P 0.5 0.3 0.1 0.2

Sediment P/A 3.1 1.2 3.3 5.9 F/P 0.1 1.5 1.2 1.0 ______

B. Bolgoda Lake Sampling sites______

WG-1 WG-2 BNL-1 BNL-2 BSL BG

Water P/A 2.4 10.5 3.3 5.4 2.4 1.0 F/P 0.6 1.2 0.5 1.0 0.6 0.9

Sediment P/A 0.4 3.5 1.0 0.4 0.4 1.5 F/P 0.1 1.3 1.0 2.4 0.0 0.0 ______

although there were exceptions (Table 3-III). For instance, the water samples collected from the BA-4 site had the P/A ratio of 14.5 and F/P ratio of 0.2, which suggested a strong input of the petrogenic PAHs. The calculated values from this application are therefore in general agreement with the method based on the ratios of 4-5 ring/2-3 ring

PAH ratios.

In addition, concentration of 1-nitropyrene was examined since the PAHs are known

to react with certain functional groups such as nitro groups (-NO2) under high temperature conditions, forming nitrated PAHs [20]. For instance, emissions from

118

combustion of fossil fuels, diesel fuels in particular, produce 1-nitropyrene and other related products. 1-Nitropyrene is potentially mutagenic and carcinogenic in humans

[13]. As shown in Table 3-I, its levels were twice the average PAHs both in the sediment and water samples from Beira Lake, particularly in samples from the BA-1,

BA-3, and BA-4 sites where the traffic flows are generally heavy and the percentage of the vehicles powered by diesel fuels is high. Thus the results appear to reflect high local inputs of 1-nitropyrene generated by motor vehicles.

3.4.4. Microtox Assay

Various tests have been developed for evaluating the toxicity of PAHs in soils and sediments, including the Microtox assays. The results are shown in (Fig. 3-4). The EC50 values for the sediments indicated that all sediments were toxic to the test organisms but that the toxicity data did not correlate well to total PAH concentrations in the sediments.

This discrepancy suggested that the toxicity was affected by the factors other than the

PAH concentration or specific PAHs present, such as particle size and the sulfur content in the sediments. From this brief study in the evaluation of toxicity by the Microtox assays, the following conclusions may be reached: the results from the short-term solid- phase assays may not be accurate in evaluating sediment toxicity and that other approaches, long-term assays should be also tried.

119

Figure 3-4. Correlation between EC50 Values obtained by Microtox assays and total PAHs for the sediments.

Acknowledgements- Thank the National Science Foundation of Sri Lanka

(RG/2001/C/05) for financial support and Dr David Hehemann and the NASA Glen

Research Center for assistance and expertise with the GC-MS.

120

3.5. References

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the environment. Environ Sci Technol 26: 1278-1284.

2. Zedeck MS (1980) Polycyclic aromatic hydrocarbons: a review. J of Envir Pathol

& Toxicol 3: 537-567.

3. USEPA Report: Assessment and Remediation of Contaminated Sediments

(ARCS) Program, Final Summary Report, EPA-905-594-001(1994), 48 pp.

4. Dahle S, Savinov VM, Matishov GG, Evenset A, Naes K (2003) Polycyclic

aromatic hydrocarbons (PAHs) in bottom sediments of the Kara Sea shelf, Gulf of

Ob and Yanisei Bay. The Science of Total Environ 306:57-71.

5. Yunker MB, Macdonald RW, Vingarzan R, Mitchell RH, Goyette D, Sylvestre S

(2002) PAHs in the Fraser River Basin: a critical appraisal of PAH ratios as

indicators of PAH source and composition. Organic Geochem 33:489-515.

6. Yunker MB, Snowdon LR, Macdonald RW, Smith JN, Fowler MG, Skibo DN,

McLaughlin FA, Danyushevskaya AJ, Petrova VI, Ivanov GI (1996) Polycyclic

aromatic hydrocarbon composition and potential sources for sediment samples

from the Beaufort and Parents Seas. Environ Sci Technol 30: 1310-1320.

7. Budzinski H, Jones I, Bellocq J. Pierard, Garrigues P (1997) Evaluation of

sediment contamination by polycyclic aromatic hydrocarbons in the Gironde

estuary. Marine Chem 58:85-97.

8. Potter TL, Simmons KE (1998) Composition of petroleum mixtures, vol 2. In:

Total Petroleum Hydrocarbon Criteria Working Group Series, Amherst Scientific

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9. Commins BT (1969) Formation of polycyclic aromatic hydrocarbons during

pyrolysis and combustion of hydrocarbons. Atmos Environ 3:565-572.

10. Wise SA, Hilpert LR, Rebbert RE, Sander LC, Schanz MM, Chesler SN, May

WE (1988) Standard reference materials for the determination of polycyclic

aromatic hydrocarbons. Fresenius A Anal Chem 332:576-582.

11. Sicre MA, Marty JC, Saliot A, Aparicio X, Grimalt J, Albaiges J (1987) Aliphatic

and aromatic hydrocarbons in different sized aerosols over the Mediterranean Sea:

Occurrence and origin. Atmos Environ 21:2247-2259.

12. IARC (International Agency for Research on Cancer (1989) Monographs and

Evaluation of the Carcinogenic Risks to Humans: In Diesel and Gasoline Engine

Exhausts and Some Nitroarene, vol. 46, IARC, Lyon, France.

13. IARC.1997. 1-nitropyrene. IARC Monograph on Evaluation of Cancer Risk of

Chemical to Humans 46:321-358.

14. O’Malley VP, Abrajano TA, Hellou J (1994) Determination of 13C/12C ratios of

individual PAHS source apportioned . Organic Geo Chem 21:809-822.

15. Neff JM. 1979. Polycyclic aromatic hydrocarbons in the aquatic environment:

sources, fates and biological effects, Applied Science Publishers Lts, Essex,

England.

16. Benner BA, Bryner NP, Wise SA, Mulholland GH, Lao RC, Fingas MF (1990)

Polycyclic aromatic hydrocarbon emissions from combustion of crude oil on

water. Environ Sci Technol 24: 1418-1427.

17. Lake JL, Norwood C, Dimock C, Bowen R (1979) Origins of polycyclic aromatic

hydrocarbons in estuarine sediments. Geochim Cosmochim Acta 43:1847-1854.

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18. Gschwend PM, Hites RA (1981) Fluxes of polycyclic aromatic hydrocarbons to

marine and lacustrine sediments in the northeastern United States. Geochim

Cosmochim Acta 45:2359-2367.

19. Albert RA, Reif AK (1988) Standard chemical thermodynamic properties of

polycyclic aromatic hydrocarbons and their isomer groups. I. Benzene series. J

Phys Chem Ref Data 17: 241-253.

20. Paputa-Peck MC, Mrano RS, Schuetzle D, Riley TC, Hampton CV, Prator TJ,

Skewes LM, Jensen TE (1983) Determination of nitrated polynuclear aromatic

hydrocarbon chromatography with nitrogen selective detection Anal Chem 55:

1946-1954.

123

APPENDICES

124

APPENDIX A

ADDITIONAL TABLES & FIGURES FOR CH I

125

(A)

LNDASHKEITDESVRVWLNSLQHLAYDIDDVLDDVATEAMHRELTPESEASTSM

VRKLIPTCCTKFSLSHRLTPKLDSINTQLQQLEKQKSDLGLIVKDEKPKGTSRRN

ETSLLESDVVGREGEKKR

(B)

Figure A-1. Tandem mass spectra of peptide (A) matched to a CC-NBS-LRR RGA type protein (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

126

(A)

(B)

Figure A-2. Tandem mass spectra of polypeptides (A,B,C) matched to Lectin (H. tuberosus) as indicated (bold) in the primary amino acid sequence (D).

127

(C)

MANNYVEVGPWGGSGGANPWSIIPNDGRITRINVRSGAIVDAIYFGYTEGGISYE

TAIFGGRNGSLSTIDIADDEEIIEINGKVGTLENLNLVSQLTFVTNKQTYGPYGT

NGGTDFSCPIAKGKVVGFFGRYGCLPGRYWCRT

(D)

Figure A-2 (cont’d). Tandem mass spectra of polypeptides (A,B,C) matched to

Lectin (H. tuberosus) as indicated (bold) in the primary amino acid sequence (D).

128

Table A-I. 1-D Hydroponic Sundance Leaf As only Marker Region Theoretical Accession Peptide kDa Mass kDa no. sp. Protein Name Score (Coverage) Peptides HQGVMVGMGQK, AVFPSIVGRPR, SYELPDGQVITIGNER, 45 42 gi|3127135 M. viride Actin 79 4 (16%) VAPEEHPILLTEAPLNPK glyceraldehyde 3-phosphate HIQAGASK, 45 44 gi|336390 Arabidopsis dehydrogenase 50 2 (5%) AVSLVLPQLK

glycolate 45 40 gi|30683149 H. annuus oxidase 24 1 (5%) IAIQAGAAGIIVSNHGAR

129

(A)

(B)

Figure A-3. Tandem mass spectra of peptides (A,B,C,D) matched to Actin (M. viride) as indicated (bold) in the primary amino acid sequence (E) in comparison to primary amino acid sequence of (H. annuus) (F).

130

(C)

(D)

Figure A-3 (cont’d). Tandem mass spectra of peptides (A,B,C,D) matched to Actin

(M. viride) as indicated (bold) in the primary amino acid sequence (E) in comparison to primary amino acid sequence of (H. annuus) (F).

131

MADEGEVSALMCDNGSGMVKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMG

QKDAYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEH

PVLLTEAPLNPKANREKMTQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDS

GDGVTHTVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVR

DIKEKLAYVAIDYEAELATANTSSSIEKSYELPDGQVITIGNERFRCPEVLFNPGY

VGMESAGIHETTYNSIMRCDVDIRKDLYGNIVLSGGSTMXPGIADRMSKEITALA

PSSMKIKVVAPPERKYSVWIGGSILASLSTFQAMWIAKSEYDESGPSIVHRKCF

(E)

MAHEGEIQPLVCDNGTGMVKAGFAGDDAPRAVFPSIVGRPRHTGVMVGMGQ

KDAYVGDEAQSKRGILTLKYPIEHGIVSNWDDMEKIWHHTFYNELRVAPEEHP

VLLTEAPLNPKANREKMTQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSG

DGVSHTVPIYKGYALPHAILRLDLAGRDLTDSLMKILTERGYMFTTTAEREIVRD

MKEKLAYVALDYEQELETAKSSSSVEKNYELPDGQVITIGAERFRCPEVLFQPS

LIGMEAAGIHETTYNSIMKCDVDIRKDLYGNIVLSGGSTMFPGIADRMSKEITALA

PSSMKIKVVAPPERKYSVWIGGSILASLSTFQQMWISKGEYDESGPSIVHRKCF

(F)

Figure A-3 (cont’d). Tandem mass spectra of peptides (A,B,C,D) matched to Actin

(M. viride) as indicated (bold) in the primary amino acid sequence (E) in comparison to primary amino acid sequence of (H. annuus) (F).

132

(A)

(B)

Figure A-4. Tandem mass spectra of peptides (A,B) matched to Glyceraldehyde 3- phosphate dehydrogenase B subunit (Arabidopsis) as indicated (bold) in the primary amino acid sequence (C) in comparison to primary amino acid sequence of

Glyceraldehyde 3-phosphate dehydrogenase (H. annuus) (D).

133

MSSIGGEASFFDAVAAQIIPKAVTTSTPVRGETVAKLKVAINGFGRIGRNFLRCW

HGRKDSPLEVVVLNDSGGVKNASHLLKYDSMLGTFKAEVKIVDNETISVDGKLI

KVVSNRDPLKLPWAELGIDIVIEGTGVFVDGPGAGKHIQAGASKVIITAPAKGAD

IPTYVMGVNEQDYGHDVANIISNASCTTNCLAPFAKVLDEEFGIVKGTMTTTHSY

TGDQRLLDASHRDLRRARAAALNIVPTSTGAAKAVSLVLPQLKGKLNGIALRVP

TPNVSVVDLVINVEKKGLTAEDVNEAFRKAANGPMKGILDVCDAPLVSVDFRCS

DVSTTIDSSLTMVMGDDMVKVVAWYDNEWGYSQRVVDLAHLVASKWPGAEA

VGSGDPLEDFCKTNPADEECKVYD

(C)

EAGAKKVLITAPGKGDIPTYVVGVNAELYNHSEAIISNASCTTNCLAPFVKVIDQ

KFGIIKGTMTTTHSYTGDQRLLDASHRDLRRARAAALNIVPTSTGAAKAVALVL

PSLKGKLNGIALRVPTPNVSVVDLVVQVSKKTFAEEVNAAFREAADNELNGILS

VCDEPLVSVDFRCSDVSSTVDSSLTMVMGDDMVKVIAWYDNEWGYSQRVVDL

ADIVANNWK

(D)

Figure A-4 (cont’d). Tandem mass spectra of peptides (A,B) matched to

Glyceraldehyde 3-phosphate dehydrogenase B subunit (Arabidopsis) as indicated

(bold) in the primary amino acid sequence (C) in comparison to primary amino acid sequence of Glyceraldehyde 3-phosphate dehydrogenase (H. annuus) (D).

134

(A)

MEITNVTEYDAIAKQKLPKMVYDYYASGAEDQWTLQENRNAFARILFRPRILID

VSKIDMTTTVLGFKISMPIMVAPTAMQKMAHPDGEYATARAASAAGTIMTLSS

WATSSVEEVASTGPGIRFFQLYVYKNRNVVEQLVRRAERAGFKAIALTVDTPRL

GRRESDIKNRFTLPPNLTLKNFEGLDLGKMDEANDSGLASYVAGQIDRTLSWKD

VQWLQTITKLPILVKGVLTGEDARIAIQAGAAGIIVSNHGARQLDYVPATISALE

EVVKATQGRIPVFLDGGVRRGTDVFKALALGASGIFIGRPVVFSLAAEGEAGVRK

VLQMLRDEFELTMALSGCRSLKEISRNHITTEWDTPRPSARL

(B)

Figure A-5. Tandem mass spectra of peptide (A) matched to Glycolate Oxidase

(Arabidopsis) as indicated (bold) in the primary amino acid sequence (B).

135

Table A-II. 1-D Sundance Leaf Hydroponic Cd, Cr, Ni, Pb Marker Region Theoretical Peptide kDa Mass kDa Accession no. sp. Protein Name Score (Coverage) Peptides 37 31 gi|126723930 H. annuus Chitinase 66 1 (3%) YGGVMoLWDR 33 kDa oxygen- 37 33 gi|22571 Arabidopsis evolving protein 51 1 (5%) GGSTGYDNAVALPAGGR

photosystem II protein < 37 38 gi|94502471 H. annuus D1 47 1 (3%) VINTWADIINR DEFIGKK, TIM Triosephosphate VASPQQAQEVHVAVR, < 37 33 gi|15226479 Arabidopsis Isomerase 45 3 (12%) IIYGGSVNGGNSAELAK hevein-like < 37 34 gi|30844170 E. europaeus antimicrobial peptide 68 1 (3%) GPLQLTWNYNYAQAGR ATP synthase CF1 < 37 55 gi|94502485 H. annuus alpha subunit 23 2 (3%) RPPGR, LIESPAPGIISR drought-induced < 37 18 gi|7489327 H. annuus protein SDi-6 31 1 (4%) SGATGATPPAEAPLVTEEK glucosyltransferase- < 37 37 gi|21594027 Arabidopsis like protein 40 1 (2%) FMTEVR

136

(A)

MEFTHPALLLLLFITVFSFLKPSTAAGIATYWGQQSDDTEGTLEAACATGNYQFV

NIAFLSTFGNNQQPVLNLAAHCDPASTCSRYSSQIKACQAQNVKVFLSIGGQRGS

YSLSSPQDAQQVADFLWNTYLGGQPATRPLGDAVLDGIDFDIEQGTDQFWSDLA

KAPAAYSSQKKVYLSAAPQCPFPSGDVRNQLLPAIREGLFDYVWVQFYNNEQCQ

YGANADALLARWNEWTQVTTNTIFLGLPAAASGAAPSGGYIPPDILTSQILPSIKS

SPKYGGVMLWDRFYDKQSGYSDAIKGSIN

(B)

Figure A-6. Tandem mass spectra of polypeptide (A) from Chitinase (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

137

(A)

MAASLQSTATFLQSAKIATAPSRGSSHLRSTQAVGKSFGLETSSARLTCSFQSDFK

DFTGKCSDAVKIAGFALATSALVVSGASAEGAPKRLTYDEIQSKTYMEVKGTGT

ANHSPTIDGGSETFSFKPGKYAGKKFCFEPTSFTVKADSVSKNAPPEFQNTKLMT

RLTYTLDEIEGPFEVASDGSVNFKEEDGIDYAAVTVQLPGGERVPFLFTVKQLDA

SGKPDSFTGKFLVPSYRGSSFLDPKGRGGSTGYDNAVALPAGGRGDEEELVKE

NVKNTAASVGEITLKVTKSKPETGEVIGVFESLQPSDTDLGAKVPKDVKIQGVW

YGQLE

(B)

Figure A-7. Tandem mass spectra of peptide (A) matched to 33 kDa Oxygen- evolving protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence (B).

138

(A)

MTAILERRESESLWGRFCNWITSTENRLYIGWFGVLMIPTLLTATSVFIIAFIAAPP

VDIDGIREPVSGSLLYGNNIISGAIIPTSAAIGLHFYPIWEAASVDEWLYNGGPYELI

VLHFLLGVACYMGREWELSFRLGMRPWIAVAYSAPVAAATAVFLIYPIGQGSFS

DGMPLGISGTFNFMIVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLI

RETTENESANEGYRFGQEEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLAAWP

VVGIWFTALGISTMAFNLNGFNFNQSVVDSQGRVINTWADIINRANLGMEVMH

ERNAHNFPLDLAAIEAPSTNG

(B)

Figure A-8. Tandem mass spectra of peptide (A) matched to photosystem II protein

D1 (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

139

(A)

(B)

Figure A-9. Tandem mass spectra of peptides (A,B,C) matched to Triosephosphate

Isomerase (TIM) (Arabidopsis) as indicated (bold) in the primary amino acid sequence (D).

140

(C)

MAATSLTAPPSFSGLRRISPKLDAAAVSSHQSFFHRVNSSTRLVSSSSSSHRSPRG

VVAMAGSGKFFVGGNWKCNGTKDSIAKLISDLNSATLEADVDVVVSPPFVYIDQ

VKSSLTDRIDISGQNSWVGKGGAFTGEISVEQLKDLGCKWVILGHSERRHVIGEK

DEFIGKKAAYALSEGLGVIACIGEKLEEREAGKTFDVCFAQLKAFADAVPSWDN

IVVAYEPVWAIGTGKVASPQQAQEVHVAVRGWLKKNVSEEVASKTRIIYGGSV

NGGNSAELAKEEDIDGFLVGGASLKGPEFATIVNSVTSKKVAA

(D)

Figure A-9 (cont’d). Tandem mass spectra of peptides (A,B,C) matched to

Triosephosphate Isomerase (TIM) (Arabidopsis) as indicated (bold) in the primary

amino acid sequence (D).

141

(A)

MKYLWVFIVFSIAVLSLACSAQQCGRQAGNRRCPNNLCCSQFGYCGRTNEYCCT

GFGCQSNCRRCGVRTVGEDVVGDIGGIISKGMFNNILKHRDDDACEGKGFYTYE

AFVAAARSFPAFGSTGDDTTRKREIAAFLAQTSHETSGGRPSAPDGPYAWGYCF

VKERNPPSKYCDTITPCPKSYYGRGPLQLTWNYNYAQAGRAIGVDLLNNPDLV

ATDAVTSFKTAIWFWMTAHSSKPSCHDVITGSWRPSASDNSVRHVPDYAVVTNI

INGEIEYGKSRNPQVEDRIEFFKRYCQILGVSPGKF

(B)

Figure A-10. Tandem mass spectra of peptide (A) matched to hevein-like antimicrobial (E. europaeus) as indicated (bold) in the primary amino acid sequence

(B).

142

(A)

(B)

Figure A-11. Tandem mass spectra of peptides (A,B) matched to ATP synthase SF1 alpha subunit (H. annuus) as indicated (bold) in the primary amino acid sequence

(C).

143

MVTIQADEISNIIRERIEQYNREVKIVNTGTVLQVGDGIARIHGLDEVMAGELVEF

QEGTIGIALNLESTNVGVVLMGDGLLIQEGSSVKATGKIAQIPVSEAYLGRVINLA

KPIDGRGEISSSEYRLIESPAPGIISRRSVYEPLQTGLIAIDSMIPIGRGQRELIIGDR

QTGKTAVATDTILNQQGQNVICVYVAIGQKASSVAQVVTTFQEKGAMEYTIVVA

ETADSPATLQYLAPYTGAALAEYFMYRERHTLIIYDDLSKQAQAYRQMSLLLRR

PPGREAYPGDVFYLHSRLLERAAKLSSLLGEGSMTALPIVETQSGDVSAYIPTNVI

SITDGQIFLSADLFNAGIRPAINVGISVSRVGSAAQIKAMKQVAGKLKLELAQFAE

LEAFAQFASDLDKATQNQLARGQRLRELLKQSQSAPLAVEEQILTIYTGTNGYLD

SLEVGQVRKFLVELRTYLKTNKPQFQEIISSTKIFTEEAEAILKEAIQEQRERFILQE

QAA

(C)

Figure A-11 (cont’d). Tandem mass spectra of peptides (A,B) matched to ATP synthase SF1 alpha subunit (H. annuus) as indicated (bold) in the primary amino acid sequence (C).

144

(A)

RGIPVRHGSRPTGKNMDSFMCNIKTVATGGHDETKTNTDQPSNTSTDLLSSAKL

VAEAAQSAATNKTDQIDKQKVAGATADLLDSSKEYGKFDESQGVGQYIKQADD

YLHKYEKSGATGATPPAEAPLVTEEKKAEAPPGVEEKGGKDESESGIGAGDAI

KAAGSFFK

(B)

Figure A-12. Tandem mass spectra of peptide (A) matched to Drought-induced protein SDi-6 (H. annuus) as indicated (bold) in the primary amino acid sequence

(B).

145

(A)

MGTPVEVSKLHFLLFPFMAHGHMIPTLDMAKLFATKGAKSTILTTPLNAKLFFEK

PIKNLNPGLEIDIQIFNFPCVELGLPEGCENVDFFTSNNNDDKNEMIVKFFFSTRFF

KDQLEKLLGTTRPDCLIADMFFPWATEAAGKFNVPRLVFHGTGYFSLCAGYCIG

VHKPQKRVASSSEPFVIPELPGNIVITEEQIIDGDGESDMGKFMTEVRESEVKSSG

VVLNSFYELEHDYADFYKSCVQKRAWHIGPLSVYNRGFEEKAERGKKANIDEAE

CLKWLDSKKPNSVIYVSFGSVAFFKNEQLFEIAAGLEASGTSFIWVVRKTKEKEE

WLPEGFEERVKGKGMIIRGWAPQVLILDHQATCGFVTHCGWNSLLEGVAAGLP

MVTWPVAAEQFYNEKLVTQVLRTGVSVGAKKNVRTTGDFISREKVVKAVREVL

VGEEADERRERAKKLAEMAKAAVEGGSSFNDLNSFIEEFTS

(B)

Figure A-13. Tandem mass spectra of peptide (A) matched to Glucosyltransferase- like protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence

(B).

146

Table A-III. 1-D Sundance Stem Hydroponic Cd, Cr, Ni, Pb Marker Region Theoretical Accession Peptide kDa Mass kDa no. sp. Protein Name Score (Coverage) Peptides Quinone oxido- 39-47 33 gi|14532287 H. annuus reductase-like 54 1 (4%) SLGADVAIDYTK phenylcoumaran benzylic ether reductase homolog VVILGDGNAK, 39-47 33 gi|7578903 T. heterophylla TH3 40 2 (3%) DKVVILGDGNAK

147

(A)

MKAWKYDEYGSVDVLKLATDVAVPEIKDDQVLVKVVAAAVNPVDYKRRLGYF

KAIDSPLPIIPGFDVLGVVLKVGSQVKDLKEGDEVYGDINDKGLEGPTQFGTLAE

YTAVEERLLALKPKNLDFIQAAALPLAIETAYEGLERAKFSEGKTILVLNGAGGV

GSFIIQLAKHVYGASKVAATSSTGKLELLKSLGADVAIDYTKENFEDLPDKYDV

VYDAIGQPEKALKAVNETGVAVSITGPIPPPGFSFILTSDGSILTKLNPYLESGKIKS

DNDPKSPFPFDKLNEAYAYLESTGPTGKVVIYPIP

(B)

Figure A-14. Tandem mass spectra of peptide (A) matched to Quinone oxido-

reductase-like protein (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

148

(A)

(B)

Figure A-15. Tandem mass spectra of peptide (A,B) matched to Phenylcoumaran benzylic ether reductase homolog (T. heterophylla) as indicated (bold) in the primary amino acid sequence (C).

149

MGSKSKILIIGATGYIGRQVAKASLALSHPTFLLVRDSPASSKPEKAQLLDSFKAS

GANILKGSLEDHASLVEAVKKVDVVISTVGGEQIANQFNIIKAIKEVGTIKRFLPSE

FGNDVDNVHAVEPAKSVFELKAQVRRAIEAESIPYTYVSSNCFAGYFLPSFAQAG

LTSPPRDKVVILGDGNAKAVYVKEEDIGTFAIKAADDPRTLNKTLYLRLPANTL

SFNELVALWEKKIGKTLEKVYVPEEHVVKLIAETPFPANIVIAIGHSIFVKGDQTN

FDIGPDGVEGSLLYPDVKYTTVDEYLSAFV

(C)

Figure A-15 (cont’d). Tandem mass spectra of peptide (A,B) matched to

Phenylcoumaran benzylic ether reductase homolog (T. heterophylla) as indicated

(bold) in the primary amino acid sequence (C).

150

Table A-IV. 1-D Sundance Leaf Hydroponic (soluble fraction) Cd, Cr, Ni, Pb Marker Region Theoretical Accession Peptide kDa Mass kDa no. sp. Protein Name Score (Coverage) Peptides VASPQQAQEVHVAVR, IIYGGSVNGGNSAELAK, 25-35 33 gi|15226479 Arabidopsis TIM 49 3 (10%) TRIIYGGSVNGGNSAELAK TRIOSEPHOSPHATE VASPAQAQEVHAGLR, 25-35 21 gi|256120 L. sativa ISOMERASE 79 2(14%) VAYALSQGLKVIACVGETLEQR

151

(A)

(B)

Figure A-16. Tandem mass spectra of peptides (A,B,C) matched to Triosephosphate

Isomerase (TIM) (Arabidopsis) as indicated (bold) in the primary amino acid sequence (D).

152

(C)

MAATSLTAPPSFSGLRRISPKLDAAAVSSHQSFFHRVNSSTRLVSSSSSSHRSPRG

VVAMAGSGKFFVGGNWKCNGTKDSIAKLISDLNSATLEADVDVVVSPPFVYIDQ

VKSSLTDRIDISGQNSWVGKGGAFTGEISVEQLKDLGCKWVILGHSERRHVIGEK

DEFIGKKAAYALSEGLGVIACIGEKLEEREAGKTFDVCFAQLKAFADAVPSWDNI

VVAYEPVWAIGTGKVASPQQAQEVHVAVRGWLKKNVSEEVASKTRIIYGGSV

NGGNSAELAKEEDIDGFLVGGASLKGPEFATIVNSVTSKKVAA

(C)

Figure A-16 (cont’d). Tandem mass spectra of peptides (A,B,C) matched to

Triosephosphate Isomerase (TIM) (Arabidopsis) as indicated (bold) in the primary amino acid sequence (D).

153

(A)

(B)

Figure A-17. Tandem mass spectra of peptides (A,B) matched to Triosephosphate

Isomerase (Arabidopsis) as indicated (bold) in the primary amino acid sequence (C).

154

IQVAAQNCWVKKGGAFTGEVSAEMLANLGVPWVILGHSERRALLNETNEFVGD

KVAYALSQGLKVIACVGETLEQREAGTTMEVVAAQTKAIADKISSWDNVVLA

YEPVWAIGTGKVASPAQAQEVHAGLRKWFCDNVSAEVSASTRIIYGGSVSGSN

CKELGGQTDVDGFLVGGASLKPEFIDIIKAAEVKKSA

(C)

Figure A-17 (cont’d). Tandem mass spectra of peptides (A,B) matched to

Triosephosphate Isomerase (Arabidopsis) as indicated (bold) in the primary amino acid sequence (C).

155

Table A-V. 1-D Sundance Leaf Soil 25-35 kDa EDTA samples Cd, Cr, Ni, Pb Theroetical Peptide EDTA Mass Accession no. sp. Protein Name score (Coverage) Peptides SSQSVKSK, ctrl 35 gi|131385 Arabidopsis OEE1 25 2 (6%) GGSTGYDNAVALPAGGR 0.1 EDTA 32 gi|126723930 H. annuus Chitinase 59 1 (2%) YGGVMLWDR 0.3 EDTA 32 gi|126723930 H. annuus Chitinase 70 1 (2%) YGGVMLWDR

156

(A)

(B)

Figure A-18. Tandem mass spectra of peptide (A,B) matched to Oxygen-evolving enhancer protein 1, chloroplast precursor (OEE1) (Arabidopsis) as indicated (bold) in the primary amino acid sequence (C).

157

MAASLQAAATLMQPTKVGGVSARNNLQLRSSQSVSKAFGLEPSASRLSCSLQTD

LKDFAQKCTDAAKIAGFALATSALVVSGANAEGVPKRLTFDEIQSKTYMEVKGT

GTANQCPTIDGGVDSFAFKPGKYNAKKFCLEPTSFTVKAEGVSKNSAPDFQKTK

LMTRLTYTLDEIEGPFEVSPDGTVKFEEKDGIDYAAVTVQLPGGERVPFLFTIKQL

VASGKPESFSVDFLVPSYRGSSFLDPKGRGGSTGYDNAVALPAGGRGDEEELQ

KENVKNTASLTGKITFTVTKSNPQTGEVIGVFESIQPSDTDLGAKTPKDVKIQGIW

YAQLES

(C)

Figure A-18 (cont’d). Tandem mass spectra of peptide (A,B) matched to Oxygen- evolving enhancer protein 1, chloroplast precursor (OEE1) (Arabidopsis) as indicated (bold) in the primary amino acid sequence (C).

158

(A)

MEFTHPALLLLLFITVFSFLKPSTAAGIATYWGQQSDDTEGTLEAACATGNYQFV

NIAFLSTFGNNQQPVLNLAAHCDPASTCSRYSSQIKACQAQNVKVFLSIGGQRGS

YSLSSPQDAQQVADFLWNTYLGGQPATRPLGDAVLDGIDFDIEQGTDQFWSDLA

KAPAAYSSQKKVYLSAAPQCPFPSGDVRNQLLPAIREGLFDYVWVQFYNNEQCQ

YGANADALLARWNEWTQVTTNTIFLGLPAAASGAAPSGGYIPPDILTSQILPSIKS

SPKYGGVMLWDRFYDKQSGYSDAIKGSIN

(B)

Figure A-19. Tandem mass spectra of polypeptide (A) from Chitinase (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

159

Table A-VI. 1-D Teddy Bear Leaf Soil 25-35 kDa EDTA samples Cd, Cr, Ni, Pb

Marker Region Theroetical Accession Peptide kDa Mass no. sp. Protein score (Coverage) Peptides

HSP70-related GVMTK, 25 - 35 28 gi|2098793 H. annuus protein 19 2 (4%) VPADVK

160

(A)

(B)

Figure A-20. Tandem mass spectra of peptides (A,B) from HSP70-related protein

(H. annuus) (C) and HSP70 (N. benthamiana) (D) as indicated (bold) in the primary amino acid sequence.

161

DLGVQGGILSGEGGDETKDILLLDVAPLTLGIETVGGVMTKLIPRNTVIPTKKSQ

VFTTYQDQQTVVSIKVYEGERSLTKDCRLLGTFDLTGIPPAPRGTPQIEVTFEVDA

NGILNVKAEDKASGKSEKITITNEKGRLSQEEIERMVREAEEFAEEDKKVKEKID

ARNALETYVYNMKNQINDKDKLADKLESDEKEKIEAATKEALDRLDDNQSAEK

EEYDEKLKEVEAVCNPIVTAVYQRSGGAPGGGAESTEDDDEHDEL

(C)

GVMTKIIPRNTTLPTSKSEVFSTAADGQTSVEINVLQGEREFVKDNKSLGRFRLD

GIPPAPRGVPQIEVKFDIDANGILSVTATDKGTGKKQDITITGASTLPKDEVDRMV

QEAEKFAKEDKEKRDSIDTKNQAESVVYQTEKQLKELGDKVPADVKDKVESKL

RELKDAISGGSTQTIKDTMAALNQEVMQLGQSLYSQPDAAAGPGPAGASGSRGK

DDGDGEVIDADFNHEFWIRYVTRLQLRDR

(D)

Figure A-20. Tandem mass spectra of peptide (A,B) from HSP70-related protein (H.

annuus) (C) and HSP70 (N. benthamiana) (D) as indicated (bold) in the primary

amino acid sequence.

162

Table A-VII. 1-D Teddy Bear Leaf Soil Cr only

Marker Region Theroetical Accession Peptide kDa Mass no. Sp. Protein Score (Coverage) Peptides cytosolic phosphoglycerate 40 42 gi|121485004 H. annuus kinase 36 1 (5%) GVTTIIGGGDSVAAVEK NILVIGPVPGQK, 200+ 35 gi|94502504 H. annuus Cytochrome f 60 2 (10%) QVVDIIPPGPELLVSEGESIK

163

(A)

MATKKSVSSLTEGDLKGKRVFVRVDLNVPLDDTFKITDDTRIRAAVPTIKYLMS

HGARVILSSHLGRPKGVTPKFSLKPLVPRLSELLGIEVKMADDCIGPDVEKLVAEI

PEGGVLLLENVRFYKEEEKNDPEFAKKLASLADLYVNDAFGTAHRAHASTEGV

AKHLKPAVAGFLVQKELDYLVGAVSNPKKPFAAIVGGSKVSSKIGVIESLLEKVD

ILVLGGGMIFTFYKAQGLAVGSSLVEEDKLDLATTLLEKAKSKGVSLLLPSDVVI

ADKFAADANSKIVPASSIPDGWMGLDIGPDSIKSFSEALDTTKTVIWNGPMGVFE

FDKFAVGTEAIAKKLAELSGKGVTTIIGGGDSVAAVEKVGLADKMSHISTGGG

ASLELLEGKPLPGVPALDEA

(B)

Figure A-21. Tandem mass spectra of peptide (A) matched to cytosolic phosphoglycerate kinase (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

164

(A)

(B)

Figure A-22. Tandem mass spectra of peptides (A,B) from cytochrome f (H. annuus) as indicated (bold) in the primary amino acid sequence (C).

165

MQTRNTFSWIKEQITRSISASLMIYIITRTSISNAYPIFAQQGYENPREATGRIVCAN

CHLANKPVDIEVPQAVLPDTVFEAVVRIPYDTQLKQVLANGKKGGLNVGAVLIL

PEGFELAPPDRISPEIKEKMGNLSFQSYRPNQKNILVIGPVPGQKYSEITFPILSPD

PATKKDIHFLKYPIYVGGNRGRGQIYPDGSKSNNTVYNATASGIVSKILRKEKGG

YEITIADASDGRQVVDIIPPGPELLVSEGESIKLDQPLTSNPNVGGFGQGDAEIVL

QDPLRVQGLLFFFAAVILAQIFLVLKKKQFEKVQLSEMNF

(C)

Figure A-22 (cont’d). Tandem mass spectra of peptides (A,B) from cytochrome f

(H. annuus) as indicated (bold) in the primary amino acid sequence (C).

166

Table A-VIII. 1-D Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb Marker Region Theoretical Accession Peptide kDa Mass kDa no. Sp. Protein Score (Coverage) Peptides malate dehydrogenase VAILGAAGGIGQPLSLLMK, 40 36 gi|2827080 M. sativa precursur 113 2 (8%) SSIEKGIKFASQ chloroplast ferredoxin-NADP+ C. oxidoreductase EPYIGR, 40 40 gi|6899972 annuum precursor 80 2 (6%) LYSIASSALGDFGDSK

167

(A)

(B)

Figure A-23. Tandem mass spectra of peptides (A,B) from malate dehydrogenase precursor (M. sativa) as indicated (bold) in the primary amino acid sequence (C).

168

MMRPSILRSVKSAVSRSITRRGYATEPVPERKVAILGAAGGIGQPLSLLMKLNP

LVSTLSLYDIAGTPGVAADVSHINSRSQVTGYAGEDELGKALEGADVVIIPAGVP

RKPGMTRDDLFNINAGIVKSLATAISKYCPHALVNMISNPVNSTVPIAAEVFKKA

GTYDEKRLFGVTTLDVVRAKTFYAGKANVPVAEVNVPVIGGHAGVTILPLFSQA

SPQANLDDDVIKALTARTQDGGTEVVTAKAGKGSATLSMAYAGAIFADACLKG

LNGVPDVVECSYVQSNLIAELPFFASKVRIGKNGVEEILGLGSLSDFEKQGLENLK

SELKSSIEKGIKFASQ

(C)

Figure A-23 (cont’d). Tandem mass spectra of peptides (A,B) from malate dehydrogenase precursor (M. sativa) as indicated (bold) in the primary amino acid sequence (C).

169

(A)

(B)

Figure A-24. Tandem mass spectra of peptides (A,B) from chloroplast ferredoxin-

NADP+ oxidoreductase precursor (C. annuum) as indicated (bold) in the primary amino acid sequence (C).

170

MATAVTAAVSLPSSKSTSFPTRTSIISPEKINFNKVPLYYRNVSGGSKLVTIRAQVT

TEAPAKVEKISKKQDEGVVVNKFRPKEPYIGRCLLNTKITGDDAPGETWHMVFS

TEGEIPYREGQSIGVIADGVDANGKPHKLRLYSIASSALGDFGDSKTVSLCVKRL

VYTNDKGEEVKGVCSNFLCDLKPGADVKITGPVGKEMLMPKDPNATVIMLGTG

TGIAPFRSFLWKMFFEKHDDYKFNGLAWLFLGVPTSSSLLYKEEFEKMKEKAPE

NFRLDFAVSREQTNEKGEKMYIQTRMAQYAEELWTLLKKDNTFVYMCGLKGM

EQGIDDIMSSLAAKEGIDWADYKKQLKKAEQWNVEVY

(C)

Figure A-24 (cont’d). Tandem mass spectra of peptides (A,B) from chloroplast ferredoxin-NADP+ oxidoreductase precursor (C. annuum) as indicated (bold) in the primary amino acid sequence (C).

171

Table A-IX. 1-D Teddy Bear Stem Hydroponic Cd, Cr, Ni, Pb

Marker Region Theoretical Accession Peptide kDa Mass kDa no. Sp. Protein Score (Coverage) Peptides

NADPH 40 34 gi|507823 Arabidopsis oxidoreductase 65 1 (3%) ILIIGGTGYLGK

malate VAILGAAGGIGQPLSLLMK, dehydrogenase ALEGADVVIIPAGVPR, 40 36 gi|2827080 M. sativa putative 48 3 (15%) DDLFNINAGIVK

172

(A)

MVVSEKSKILIIGGTGYIGKYLVETSAKSGHPTFALIRESTLKNPEKSKLIDTFKS

YGVTLLFGDISNQESLLKAIKQVDVVISTVGGQQFTDQVNIIKAIKEAGNIKRFLPS

EFGFDVDHARAIEPAASLFALKVRIRRMIEAEGIPYTYVICNWFADFFLPNLGQLE

AKTPPRDKVVIFGDGNPKAIYVKEEDIATYTIEAVDDPRTLNKTLHMRPPANILSF

NEIVSLWEDKIGKTLEKLYLSEEDILQIVQEGPLPLRTNLAICHSVFVNGDSANFE

VQPPTGVEATELYPKVKYTTVDEFYNKFV

(B)

Figure A-25. Tandem mass spectra of peptides (A) matched to NADPH oxidoreductase, putative (Arabidopsis) as indicated (bold) in the primary amino acid sequence (B).

173

(A)

(B)

Figure A-26. Tandem mass spectra of peptides (A,B,C) matched to malate dehydrogenase precurser (M. sativa) as indicated (bold) in the primary amino acid sequence (D).

174

(C)

MMRPSILRSVKSAVSRSITRRGYATEPVPERKVAILGAAGGIGQPLSLLMKLNP

LVSTLSLYDIAGTPGVAADVSHINSRSQVTGYAGEDELGKALEGADVVIIPAGVP

RKPGMTRDDLFNINAGIVKSLATAISKYCPHALVNMISNPVNSTVPIAAEVFKKA

GTYDEKRLFGVTTLDVVRAKTFYAGKANVPVAEVNVPVIGGHAGVTILPLFSQA

SPQANLDDDVIKALTARTQDGGTEVVTAKAGKGSATLSMAYAGAIFADACLKG

LNGVPDVVECSYVQSNLIAELPFFASKVRIGKNGVEEILGLGSLSDFEKQGLENLK

SELKSSIEKGIKFASQ

(D)

Figure A-26 (cont’d). Tandem mass spectra of peptides (A,B,C) matched to malate dehydrogenase precurser (M. sativa) as indicated (bold) in the primary amino acid sequence (D).

175

Table A-X. 1-D Teddy Bear Leaf Hydroponic (Soluble fraction) Cd, Cr, Ni, Pb

Marker Region Theoretical Peptide kDa Mass kDa Accession no. Sp. Protein Score (Coverage) Peptides

25-35 32 gi|126723930 H. annuus Chitinase 49 1 (3%) YGGVMLWDR

115-125 106 gi|3415117 Arabidopsis villin 3 28 1(2%) AAALAALTSAFNSSSGR

176

(A)

MEFTHPALLLLLFITVFSFLKPSTAAGIATYWGQQSDDTEGTLEAACATGNYQFV

NIAFLSTFGNNQQPVLNLAAHCDPASTCSRYSSQIKACQAQNVKVFLSIGGQRGS

YSLSSPQDAQQVADFLWNTYLGGQPATRPLGDAVLDGIDFDIEQGTDQFWSDLA

KAPAAYSSQKKVYLSAAPQCPFPSGDVRNQLLPAIREGLFDYVWVQFYNNEQCQ

YGANADALLARWNEWTQVTTNTIFLGLPAAASGAAPSGGYIPPDILTSQILPSIKS

SPKYGGVMLWDRFYDKQSGYSDAIKGSIN

(B)

Figure A-27. Tandem mass spectra of polypeptide (A) from Chitinase (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

177

(A)

MSGSTKVLDPAFQGVGQKPGTEIWRIENFEPVPVPKSEHGKFYMGDTYIVLQTTQNKGGAYLFDI

HFWIGKDTSQDEAGTAAVKTVELDAALGGRAVQYREIQGHESDKFLSYFKPCIIPLEGGVASGFKK

PEEEEFETRLYTCKGKRAVHLKQVPFARSSLNHDDVFILDTKEKIYQFNGANSNIQERAKALVVIQ

YLKDKFHEGTSDVAIVDDGKLDTESDSGEFWVLFGGFAPIARKVASEDEIIPETTPPKLYSIADGQV

ESIDGDLSKSMLENNKCYLLDCGSEIFIWVGRVTQVEERKTAIQAAEDFVASENRPKATRITRVIQG

YEPHSFKSNFDSWPSGSATPANEEGRGKVAALLKQQGVGLKGLSKSTPVNEDIPPLLEGGGKLEV

WYIDANSKTVLSKDHVGKLYSGDCYLVLYTYHSGERKEDYFLCCWFGKNSNQEDQETAVRLAST

MTNSLKGRPVQARIFEGKEPPQFVALFQHMVVLKGGLSSGYKNSMTEKGSSGETYTPESIALIQVS

GTGVHNNKALQVEAVATSLNSYDCFLLQSGTSMFLWVGNHSTHEQQELAAKVAEFLKPGTTIKH

AKEGTESSSFWFALGGKQNFTSKKVSSETVRDPHLFSFSFNRGKFQVEEIHNFDQDDLLTEEMHLL

DTHAEVFVWVGQCVDPKEKQTAFEIGQRYINLAGSLEGLSPKVPLYKITEGNEPCFFTTYFSWDST

KATVQGNSYQKKAALLLGTHHVVEDQSSSGNQGPRQRAAALAALTSAFNSSSGRTSSPSRDRSN

GSQGGPRQRAEALAALTSAFNSSPSSKSPPRRSGLTSQASQRAAAVAALSQVLTAEKKKSPDTSPS

AEAKDEETSFSEVEATEEATEAKEEEEVSPAAEASAEEAKPKQDDSEVETTGVTFTYERLQAKSEK

PVTGIDFKRREAYLSEVEFKTVFGMEKESFYKLPGWKQDLLKKKFNLF (B)

Figure A-28. Tandem mass spectra of peptide (A) matched to villin 3 (Arabidopsis) as indicated (bold) in the primary amino acid sequence (B).

178

Table A-XI. 1-D Teddy Bear Leaf Hydroponic (Transmembrane fraction) Cd, Cr, Ni, Pb

Marker Region Theoretical Accession Peptide kDa Mass kDa no. Sp. Protein Score (Coverage) Peptides

photosystem II 35 38 gi|94502471 H. annuus protein D1 28 1 (3%) VINTWADIINR,

179

(A)

MTAILERRESESLWGRFCNWITSTENRLYIGWFGVLMIPTLLTATSVFIIAFIAAPP

VDIDGIREPVSGSLLYGNNIISGAIIPTSAAIGLHFYPIWEAASVDEWLYNGGPYELI

VLHFLLGVACYMGREWELSFRLGMRPWIAVAYSAPVAAATAVFLIYPIGQGSFS

DGMPLGISGTFNFMIVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLI

RETTENESANEGYRFGQEEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLAAWP

VVGIWFTALGISTMAFNLNGFNFNQSVVDSQGRVINTWADIINRANLGMEVMH

ERNAHNFPLDLAAIEAPSTNG

(B)

Figure A-29. Tandem mass spectra of peptide (A) matched to photosystem II protein D1 (H. annuus) as indicated (bold) in the primary amino acid sequence (B).

180

Table A-XII. 2-D Teddy Bear Leaf Hydroponic Cd, Cr, Ni, Pb Marker Region Theoretical Accession Peptide kDa Mass kDa no. sp Protein Score (Coverage) Peptides RPHAVLINSISSSR, M. Cytokinin-O- NFRLKDLPRLIK, 25-35 31 gi|92884215 truncatula glucosyltransferase 15 3 (8%) NPNDLTIRFNTEVADKCLR ABA/WDS stress 25-30 24.5 gi|38605916 O. sativa induced 17 2 (4%) KHHLFG, AQKPVHDASKR

NAC Domain GRGLLVGMK, 25-30 34 gi|51963656 O. sativa protein 18 2 (4%) GEKTPWMHEYR similar to 1- aminocyclopropane- 1-carboxylic acid DFQDAIK, VYGPIK, 45 42 gi|15221544 Arabidopsis oxidase 16 3 (4%) GLGGTSALLDFRV 1- Rosa aminocyclopropane- hybrid 1-carboxylic acid 45 41 gi|20372648 cultivar oxidase 27 1 (2%) AIANFMSKAR

181

(A)

(B)

Figure A-30. Tandem mass spectra of peptides (A,B,C) matched to Cytokinin-O- glucosyltransferase (M. truncatula) as indicated (bold) in the primary amino acid sequence (D).

182

(C)

MTNFEEKRPHAVLINSISSSRGENAFDGFTDFNFETIPDGLTPKDGDGDISQDLHS

LGESIITNFHHFFDELLAKLQDSATAGLIPPVTCLVSDCYMPFTVAAAEEHALPVV

LFSPSRACYFLACLLSPKMYQNSQLPFKDESDLTNEYLDTKVDWIPGLKNFRLK

DLPRLIKTKNPNDLTIRFNTEVADKCLRASGMVFNTSNELESDVMNAFYSMFP

SLYTIGPLASFVNQSPQNHLTSLDCNLWKEDTKCLEWLESKEPGSVVYVNFGSIT

VKSQEKP

(D)

Figure A-30 (cont’d). Tandem mass spectra of peptides (A,B,C) matched to

Cytokinin-O-glucosyltransferase (M. truncatula) as indicated (bold) in the primary

amino acid sequence (D).

183

(A)

(B)

Figure A-31. Tandem mass spectra of peptides (A,B) matched to ABA/WDS stress induced (O. sativa) as indicated (bold) in the primary amino acid sequence (C).

184

MADEYGRGGYGRSGAGAGDDYESGGYNRSSSGGADEYAAGRSGRAQKPVXD

ASKRFTKSRRRATTYGXRRRRVNKSGPRASDSGXNNRSGANRSTAATSPARRV

QRRXGAEADEEYVDGLSSRAPGEVQEGGEGAQEQGAPREVGPXRRXFAMYERH

QAKKDPENAQRHRIEEGVAAAAALGSGGFAFHEHHDKKEAKQAAKDAEEEAEE

ESGSGARGGEGKKKHHLFG

(C)

Figure A-31(cont’d). Tandem mass spectra of peptides (A,B) matched to ABA/WDS stress induced (O. sativa) as indicated (bold) in the primary amino acid sequence(C).

185

(A)

MSESEVSVINQLEEEETRLELPPGFRFHPTDEEVVTHYLTRKAQDRSFSCVVIADV

NLNNCEPWDLPSKAKMGEKEWFFFCHKDRKYPTGMRTNRATASGYWKATGKD

KEIFRGRGLLVGMKKTLVFYMGRAPRGEKTPWVMHEYRLDGKLPPNLPRSA

KEEWAVCRVFNKDLAAKIAQMPPPPFPRNDSFDLDLDDFLHLDADLPPLIDDPFA

STSTLKTEPPPPANLMHNHYGYFSLPASATNYNHSSGAMADQAIRRFCKAEAST

ACFSGADADVDPVVDELLSFPDSITDYSYIWKA

(B)

Figure A-32. Tandem mass spectra of peptide (A) matched to NAC Domain protein

(O. sativa) as indicated (bold) in the primary amino acid sequence (B).

186

(A)

(B)

Figure A-33. Tandem mass spectra of peptides (A,B,C) matched to similar to 1- aminocyclopropane-1-carboxylic acid oxidase (Arabidopsis) as indicated (bold) in the primary amino acid sequence (D).

187

(C)

MESTKIAPSFDRASELKAFDETKTGVKGLVDSGISKIPRIFHHSSVELANPKPLPSD

LLHLKTIPTIDLGGRDFQDAIKHKNAIEGIKEAAAKWGFFQVINHGVSLELLEKM

KDGVRDFHEQPPEVRKDLYSRDFGRKFIYLSNFDLYTAAAANWRDTFYCYMAP

DPPEPQDLPEICRDVMMEYSKQVMILGEFLFELLSEALGLNPNHLKDMECLKGL

RMLCHYFPPCPEPDLTFGTSKHSDGSFLTVLLPDNIEGLQVCREGYWFDVPHVPG

ALIINIGDLLQLITNDKFISLKHRVLANRATRARVSVACFFHTHVKPNPRVYGPIK

ELVSEENPPKYRETTIRDYATYFNGKGLGGTSALLDFKV

(D)

Figure A-33 (cont’d). Tandem mass spectra of peptides (A,B,C) matched to similar to 1-aminocyclopropane-1-carboxylic acid oxidase (Arabidopsis) as indicated (bold) in the primary amino acid sequence (D).

188

(A)

MGLAENQLSFDVIEEWIKKNPKASICTPEGVAEFKNVANFQDYHGFPDFRKAIAN

FMSKARGGRVKFDPDRVVMAGGATGANETVMFCLADPGDAFLVPSPYYPAFY

RDLGWRTGVKIVPVDCDSSNNFQVTRAALEAAYEKARNNNINIKGLIITNPSNPL

GTTVDRDTLTSLVKFINDKNIHLVCDEIYAGTVFSCPKFTCVTEVLQDVKNCNRD

LIHIVYSLSKDMGVPGLRIGIVYSYNDAVVNCARKMSSFGLVSSQTQHMLSAML

LDDEFVSNFLETSSKRLARRHRFFTTGLEEVGINCLKGNAGLYCWMDLRKLLKD

QTFEAEMVLWRMIINEVKLNVSPGSSFRCVEPGWFRVC

(B)

Figure A-34. Tandem mass spectra of peptide (A) from 1-aminocyclopropane-1- carboxylic acid oxidase (Rosa hybrid cultivar) as indicated (bold) in the primary amino acid sequence (B).

189

(A)

MAVVMKVGAWGAPDGSPQDINAESRPQRLESITIYSVESPGVCGIKGFSFKYVD

QQGSSVKSAIWGSNSGNPNTIEMREGEQLKLVGGTFDNEGIGSLTLETNTTKHKT

YGYPVQGGEFSLPLPQGKGELVAFFGRSDVTLKALGVYVKGSPAKVGKWGAK

SGAPRDIRPDADPCKLESFTIHSSERIHGFSFTYLTKSGQAISVPLWGKKAGEEHTI

FMNQSEYVSSITGAYDTYGITFLNFDTNQGASHTFGRNPSAGTKTFSVPLPDNGA

LDDAAAVAFFGSSGDSLVAIGAYVGVAPE

(B)

Figure A-35. Tandem mass spectra of a polypeptide (A) matched to Jacalin-like protein LEM2 (H. vulgare) as indicated (bold) in the primary amino acid sequence

(B).

190

(A)

MKYLWVFIVFSIAVLSHACSAQQCGRQAGNRRCANNLCCSQYGYCGRTNEYCC

TSQGCQSQCRRCGVRTVGEIVVGDIGGIISKGMFNNILKHRDDDACEGKGFYTYE

AFVAAARSFPAFGSTGDDATRKREIAAFLAQTSHETSAGWPSAPDGPYAWGYCF

VRERNPPSKYCDTTTPCPKSYYGRGPIQLTWNYNYEQAGRAIGADLLNNPDLVA

TDAVISFKTAIWFWMTAQSSKPSCHDVITGSWRPSASDNSVCHVPDYAVVTNIIS

GEIEYGKSRNPQVEDRIEFFKRYCQILGVSPGKCYEERTFVSGLMMETI

(B)

Figure A-36. Tandem mass spectra of polypeptide (A) matched to hevein-like antimicrobial protein (E. europaeus) as indicated (bold) in the primary amino acid sequence (B).

191

(A)

(B)

Figure A-37. Tandem mass spectra of polypeptides (A,B,C,D,E,F,G) matched to

HSP 70 protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence (H).

192

(C)

(D)

Figure A-37 (cont’d). Tandem mass spectra of polypeptides (A,B,C,D,E,F,G) matched to HSP 70 protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence (H).

193

(E)

(F)

Figure A-37 (cont’d). Tandem mass spectra of polypeptides (A,B,C,D,E,F,G) matched to HSP 70 protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence (H).

194

(G)

MATKSEKAIGIDLGTTYSCVGVWMNDRVEIIPNDQGNRTTPSYVAFTDTERLIGDAAKNQVALN

PQNTVFDAKRLIGRKFSDPSVQSDILHWPFKVVSGPGEKPMIVVSYKNEEKQFSPEEISSMVLVKM

KEVAEAFLGRTVKNAVVTVPAYFNDSQRQATKDAGAISGLNVLRIINEPTAAAIAYGLDKKGTK

AGEKNVLIFDLGGGTFDVSLLTIEEGVFEVKATAGDTHLGGEDFDNRLVNHFVAEFRRKHKKDIA

GNARALRRLRTACERAKRTLSSTAQTTIEIDSLHEGIDFYATISRARFEEMNMDLFRKCMDPVEK

VLKDAKLDKSSVHDVVLVGGSTRIPKIQQLLQDFFNGKELCKSINPDEAVAYGAAVQAAILTGEG

SEKVQDLLLLDVAPLSLGLETAGGVMTVLIPRNTTVPCKKEQVFSTYADNQPGVLIQVYEGERAR

TRDNNLLGTFELKGIPPAPRGVPQINVCFDIDANGILNVSAEDKTAGVKNQITITNDKGRLSKEEIE

KMVQDAEKYKAEDEQVKKKVEAKNSLENYAYNMRNTIKDEKLAQKLTQEDKQKIEKAIDETIE

WIEGNQLAEVDEFEYKLKELEGICNPIISKMYQGGAAAGGMPTDGDFSSSGAAGGPKIEEVD

(H)

Figure A-37 (cont’d). Tandem mass spectra of polypeptides (A,B,C,D,E,F,G) matched to HSP 70 protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence (H).

195

(A)

MISYVDIQQMTLHLIMKIPSTISREHGKLYKFRRIQQYIKTSMDSFLKVTYLIFIVV

FYIRVSSKGAYAMANLSPPVRTCVLRVGIKCCKGCQTKAKRKLLNVSGVSTVE

YNAEQGLLTVTGDANPTTLLHKLTKWGKKAELVSFLGDNYSSHVPRTPEQNQN

KTMEKKKKKPTKCCLLMCFGNKRSKNTKIEPMAIPNWQYRGVGNENGNARPFI

NAAMSPPMVYPPPQAVPGFTTPIPYPPPSFFPGRPPPPYTGAGMFQSAPPQSPPYFP

VPNPRLHYPHH

(B)

Figure A-38. Tandem mass spectra of polypeptide (A) matched to metal ion binding protein (Arabidopsis) as indicated (bold) in the primary amino acid sequence (B).

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APPENDIX B

ADDITIONAL FIGURES FOR CH II

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Figure B-1. Chromatographic apparatus designed for whole protein separation.

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Figure B-2. Column switch diagram of offline cap-trap enrichment and online sample introduction.

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Figure B-3. Chromatographic apparatus designed for small molecule/peptide separation with post column split.

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2.5

1.5 No TPCK CHY+TPCK 10A CHY+TPCK 30A CHY+TPCK 10 B Absorbance CHY + TPCK 30 B 1

0.5

0 0 200 400 600 800 1000 1200 1400 Time (s)

Figure B-4. UV spectrum analysis of CHY and CHY-TPCK samples enzymatic activity by reaction with BTEE observed at 256 nm.

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